CN109312386B

CN109312386B - Method of screening target-specific nucleases using a multi-target system of in-target and off-target targets and uses thereof

Info

Publication number: CN109312386B
Application number: CN201780037444.4A
Authority: CN
Inventors: 李政埈
Original assignee: Toolgen Inc
Current assignee: Toolgen Inc
Priority date: 2016-06-15
Filing date: 2017-06-14
Publication date: 2022-10-25
Anticipated expiration: 2037-06-14
Also published as: WO2017217768A1; CN109312386A; KR102523302B1; EP3473728A4; EP3473728A1; JP6940117B2; KR20190008548A; EP3473728B1; US20190177710A1; JP2019517802A

Abstract

The present invention relates to a method of selecting target-specific nucleases having high specificity and high activity, and more particularly, to a method of screening and selecting a target-specific nuclease system by using a multiple target system capable of recognizing off-target activity and on-target activity simultaneously. By the present invention, a target-specific nuclease with reduced off-target effect and improved on-target effect can be selected.

Description

Method for screening target-specific nucleases using a multiple target system of in-target and off-target targets and uses thereof

Technical Field

The present invention relates to a method of selecting target-specific nucleases having high specificity and high activity, and more particularly, to a method of screening and selecting a target-specific nuclease system by using a multiple target system capable of recognizing off-target activity and on-target activity simultaneously.

Background

The CRISPR-Cas system consists of a guide RNA having a sequence complementary to a gene or nucleic acid to be targeted and a CRISPR enzyme, which is an enzyme that cleaves the gene or nucleic acid to be targeted, the guide RNA and the CRISPR enzyme forming a CRISPR complex by means of which the targeted gene or nucleic acid is cleaved or modified.

However, an unsolved problem is that a non-targeted gene or nucleic acid is modified while the above-described effect of modifying a gene or nucleic acid to be targeted is produced.

Non-targeted genes or nucleic acids can partially interact with the guide RNA by including sequences that are partially complementary to the guide RNA, by which the CRISPR complex cleaves or modifies the corresponding gene (i.e., the undesired gene or nucleic acid). This effect is called off-target effect.

Therefore, it is important to increase the on-target effect to increase the efficiency of modifying a target gene or a target nucleic acid by using the CRISPR-Cas system, or to reduce or eliminate the off-target effect of the CRISPR-Cas system to solve problems such as genetic defects that may be caused because an undesired gene or nucleic acid is modified. Furthermore, with the reduction of the above-mentioned off-target effects of the CRISPR-Cas system, it can be expected that the gene or nucleic acid to be targeted can be precisely modified with accuracy and specificity.

To reduce or eliminate off-target effects and/or improve the accuracy and specificity of the CRISPR-Cas system, the following aspects are considered to be important parts: selecting (i.e., selecting targets with low off-target effects) or modifying guide RNAs with low off-target effects and modulating activity (or efficiency) and specificity of CRISPR enzymes.

In order to modulate the activity or specificity of CRISPR enzymes, various strategies were tried and developed, such as methods of modulating Cas9 concentration in cells or causing single-stranded DNA fragmentation using Cas9 nickase, methods of fusion proteins generated by fusing fokl nuclease domains with catalytically inactive Cas9, and the use of truncated guide RNAs with partial sequences of the 5' end of the guide RNA removed.

Accordingly, the present inventors have developed a screening method for selecting a target-specific nuclease having high specificity and/or activity, and confirmed that an excellent target-specific nuclease can be efficiently selected from a candidate group of a plurality of modified nucleases using the screening method, thereby completing the present invention.

Documents of the prior art

Patent document

WO 2013-098244

WO 2015-123339

WO 2014-093661

WO 2014-204724

KR 10-2016-0058703

Non-patent document

1.Anders，C.et al..2014.Structural basis of PAM-dependent target DNA recognition by the Cas9endonuclease.Nature 513(7519)，569-573.

2.Anderson，E.M.，et al.，2015.Systematic analysis of CRISPR？Cas9mismatch tolerance reveals low levels of off-target activitv.J.Biotechnol.211，56-65.

3.Frock，R.L.et al.，2015.Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases.Nat.Biotechnol.33(2)，179-186

4.Bauer，D.E.et al.，2015.Generation of genomic deletions in mammalian cell lines via CRISPR/Cas9.J.Vis.Exp.95.

5.Brinkman，E.K.et al.，2014.Easy quantitative assessment of genome editing by sequence trace decomposition.Nucleic Acids Res.42(22)，e168.

6.Cho，S.W.et al.，2014.Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and

(nickase)s.Genome Res.24(1)，132-141.

7.Christian，M.et al.2010.Targeting DNA double-strand breaks with TAL effector nucleases.Genetics 186(2)，757-761.

8.Cradick，T.J.et al.，2013.CRISPR/Cas9systems targeting β-globin and CCR5genes have substantial off-target activity.Nucleic Acids Res.41(20)，9584-9592.

9.Crosetto，N.et al.，2013.Nucleotide-resoluIion DNA double-strand break mapping by next-generation sequencing.Nat.Methods 10(4)，361-365.

10.Dahlem，T.J.et al.，2012.Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome.PLoS Genet.8(8)，e1002861.

11.Doench.J.G.et al.，2016.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9.Nat.Biotechnol.34(2)，184-191

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Disclosure of Invention

Technical problem

It is an object of the present invention to provide a screening system for selecting target-specific nucleases using multiple targets comprising one or more on-targets and one or more off-targets.

It is another object of the invention to provide a selection system for target-specific nucleases using multiplexed targets comprising one or more in-target targets and one or more off-target targets.

It is a further object of the invention to provide kits or compositions for screening and/or selecting target-specific nucleases using multiple targets.

It is a further object of the invention to provide a cell comprising multiple targets for screening and/or selecting target-specific nucleases and/or a method of producing the cell.

It is yet another object of the present invention to provide a system for screening and selecting target-specific nucleases using multiple targets and the overall use of the nucleases selected using this system.

Technical scheme

To solve the problem, the present invention provides a screening system for selecting a target-specific nuclease having high specificity or high activity, and various uses of the screening system.

The target-specific nuclease may comprise a moiety capable of specifically recognizing the target and a moiety capable of cleaving or modifying the recognized target, and the moiety capable of specifically recognizing the target and/or the moiety capable of cleaving or modifying the recognized target may be screened and/or selected by means of the screening system. Screening system

The term "screening system for selecting target-specific nucleases" as used herein refers to a concept that includes all of the following: kits and compositions for selecting target-specific nucleases with high specificity and activity, methods of using the same, and various intermediates derived in the process.

Thus, the invention described as a "system" in this specification may be construed as a composition or method to suit the aspects of the respective invention, as long as the invention is used to select the desired target-specific nuclease.

One aspect of the invention relates to methods for screening target-specific nuclease systems and compositions for use in the methods.

Another aspect of the present invention relates to a method for selecting a target-specific nuclease having high specificity or high activity by means of the screening method and a composition for use in the method.

As a preferred embodiment, the present invention provides:

a screening system or composition for selecting target-specific nucleases having high specificity or high activity, the screening system or composition comprising:

i) A nuclease comprising a moiety capable of recognizing a target and a moiety capable of cleaving or modifying the recognized target; and

ii) a multiplexed target comprising one or more selection elements, wherein the multiplexed target comprises one or more in-target targets and one or more off-target targets.

As another preferred embodiment, the present invention provides:

ii) a multiplexed target comprising one or more selection elements, wherein the multiplexed target comprises one in-target and one or more off-target targets.

As a further preferred embodiment, the present invention provides:

ii) a multiplexed target comprising a selection element, wherein the multiplexed target comprises an in-target and an off-target.

The screening system can be used to select excellent target-specific nucleases with high specificity and/or high activity from a variety of target candidate sets.

Nuclease enzymes

The term "target-specific nuclease" refers to a nuclease that is capable of recognizing and cleaving a specific location of a nucleic acid (DNA or RNA) in a desired (target or targeted) genome. In the present invention, the target-specific nuclease is also used as a concept including other elements necessary for allowing the specific nuclease to perform a desired function.

The nuclease may include a nuclease fused with a domain (portion) capable of recognizing a specific target sequence in a genome and a domain (portion) capable of cleaving it, respectively, and examples thereof may include, but are not limited to: meganucleases; a fusion protein in which a transcription activator-like (TAL) effector domain (which is a transcription activator-like effector nuclease (TALEN) derived from a plant-pathogenic gene, and is a domain capable of recognizing a specific target sequence in a genome) and a cleavage domain are fused; zinc finger nucleases; or an RNA-guided engineered nuclease (RGEN). RGEN may be preferably used in the present invention.

In the present invention, "RGEN" includes the CRISPR-Cas system, which consists of a target-specific guide RNA and a CRISPR enzyme.

The "CRISPR-Cas system" of the present invention comprises sequences encoding a guide RNA and a CRISPR enzyme and comprises all elements involved in inducing its expression or activity. The CRISPR-Cas system can be a vector system comprising sequences encoding a guide RNA and a CRISPR enzyme.

The constituent elements contained in the vector system are operably linked to each other. The term "operably linked" refers to a functional linkage between nucleic acid sequences such that the sequences encode a desired function. For example, when the linked promoter and/or regulatory region functionally regulates expression of the coding sequence, the desired gene (e.g., the coding sequence of the selectable marker) is in operable linkage with its promoter and/or its regulatory sequence. This refers to the linkage between coding sequences, whereby these sequences may also be regulated by the same linked promoter and/or regulatory region, and this linkage between coding sequences may also refer to the linkage in reading frame or at the same coding frame. The term "operably linked" also refers to a linkage of sequences that are functional but not coding (e.g., self-amplifying sequences or origins of replication). These sequences are in an operably linked state when they are capable of performing their normal function (e.g., when a vector comprising these sequences in a host cell is capable of replication, amplification and/or isolation).

Furthermore, the "CRISPR-Cas system" of the present invention, which may be a Ribonucleoprotein (RNP) system in which the guide RNA and the CRISPR enzyme form a complex, comprises the sequence of the guide RNA and the protein or polypeptide of the CRISPR enzyme and comprises all elements involved in inducing its expression or activity. In this case, RNP is a protein-RNA complex, and refers to a form of a complex formed by interaction of RNA and an RNA-binding protein.

The "CRISPR enzyme" of the present invention is the main protein component of the CRISPR-Cas system, which forms a complex with a guide RNA, thereby forming an active CRISPR-Cas system or an inactive CRISPR-Cas system.

In this case, an inactive CRISPR-Cas system refers to a functionally inactive CRISPR-Cas system of all or part of the contained CRISPR enzymes; and, the CRISPR-Cas system, whose functional part is inactive, can be used as a nickase for cleaving single-stranded nucleic acids.

As an example, when the CRISPR enzyme is Cas9, the CRISPR enzyme can be prepared by introducing the mutation D10A or H840A into the Cas9 protein; when bound to guide RNA, the D10A Cas9 protein, H840A Cas9 protein (i.e., a Cas9 protein prepared by introducing mutations into only one active site of the Cas9 protein) can act as a nickase.

As another example, when an inactive Cas9 protein binds to a guide RNA, a D10A/H840A Cas9 protein prepared by introducing both mutations D10A and H840A into the Cas9 protein, i.e., an inactive Cas9 protein (dead Cas9, dCas 9) prepared by introducing mutations into both active sites of the Cas9 protein, can form a nucleic acid binding complex without cleaving the target gene or nucleic acid sequence.

CRISPR enzymes are nucleic acids having a sequence encoding a CRISPR enzyme or polypeptide (or protein), typically a type II CRISPR enzyme or a type V CRISPR enzyme is commonly used.

Examples of type II CRISPR enzymes include Cas9, and Cas9 may be Cas9 derived from a variety of microorganisms, such as actinomyceta (actinobacilla) (e.g., actinomycete naeslundii) Cas9; water producing phylum (Aquificae) Cas9; bacteroidetes (bacteroidides) Cas9; chlamydomonas (Chlamydiae) Cas9; curvularia viridis (Chloroflexi) Cas9; cyanobacteria (Cyanobacteria) Cas9; phylum traceobacterium (elsusimicrobia) Cas9; phylum fibrobacter (fibrobacter) Cas9; firmicutes Cas9 (e.g., streptococcus pyogenes Cas9, streptococcus thermophilus Cas9, listeria innocua (Listeria innocus) Cas9, streptococcus agalactiae (Streptococcus agalactiae) Cas9, streptococcus mutans (Streptococcus mutans) Cas9, and Enterococcus faecium (Enterococcus faecalis) Cas 9); fusobacteria (fusobacteriacea) Cas9; proteobacteria (Proteobacteria) (e.g., neisseria meningitidis (Neisseria meningitidis), campylobacter jejuni (Campylobacter jejuni)) Cas9, and spirochaeta (e.g., treponema pallidum) Cas9.

The "Cas9" is an enzyme capable of cleaving or modifying the sequence or position of a gene or nucleic acid to be targeted by binding to a guide RNA, and may be composed of an HNH domain (capable of cleaving a nucleic acid strand that binds complementary to the guide RNA), a RuvC domain (capable of cleaving a nucleic acid strand that binds non-complementary to the guide RNA), a REC domain (capable of recognizing a target), and a PI domain (capable of recognizing PAM). For specific structural features of Cas9, reference may be made to Hiroshi Nishimasu et al, (2014) Cell 156:935-949.

Further, examples of type V CRISPR enzymes include Cpf1, and Cpf1 may be Cpf1 derived from a microorganism: streptococcus (Streptococcus), campylobacter (Campylobacter), nitratifractor, staphylococcus (Staphylococcus), parvibacterium, roseburia (Roseburia), neisseria (Neisseria), acetobacter gluconicum (Gluconobacter), azospirillum, sphaechaeta Lactobacillus (Lactobacillus), eubacterium (Eubacterium), corynebacterium (Corynebacterium), carnobacterium (Carnobacterium), rhodobacterium (Rhodobacterium), listeria (Listeria), paludibacter, clostridium (Clostridium), mao Luojun (Lachnospiraceae), clostridium cilium (Leptotrichia), francisella (Francisella), legionella (Legionella), alicyclobacillus (Alicyclobacillus), methylophilus methanophilus (Methanomethyphyllius), porphyromonas (Porphyromonas), prevotella (Prevotella), bacteroides (Bacteroides), streptococcus (Helcococcus), letospora, vibrio Desulfovibrio (Desulfovibrio), desulfonitronum, feng Youjun (Opituceae), tuberibacillus, bacillus (Bacillus), brevibacterium (Brevibacterium), methylobacterium (Methylobacterium), or Aminococcus (Acidaminococcus).

Examples of Cpf1 include a similar RuvC domain corresponding to the RuvC domain of Cas9, lacking the HNH domain of Cas9 and comprising a Nuc domain; cpf1 may consist of a REC domain, a WED domain (capable of recognizing a target) and a PI domain (capable of recognizing PAM). For specific structural features of Cpf1, reference may be made to Takashi Yamano et al, (2016) Cell 165:949-962.

CRISPR enzymes can recognize Protospacer (PAM) proximity motifs in genes or nucleic acid sequences, and PAM can vary depending on the type and/or source of the CRISPR enzyme.

For example, when the CRISPR enzyme is SpCas9, the PAM can be 5'-NGG-3'; when the CRISPR enzyme is StCas9, the PAM can be 5'-NNAGAAW-3' (W = a or T); when the CRISPR enzyme is NmCas9, the PAM may be 5'-NNNNGATT-3'; when the CRISPR enzyme is CjCas9, the PAM can be 5'-NNNVRYAC-3' (V = G or C or a, R = a or G, Y = C or T), in which case N can be A, T, G or C; or A, U, G or C. Furthermore, when the CRISPR enzyme is FnCpf1, the PAM may be 5'-TTN-3'; when the CRISPR enzyme is AsCpf1 or LbCpf1, the PAM can be 5'-TTTN-3', in which case N can be A, T, G or C; or A, U, G or C.

Information on CRISPR enzymes is available from publicly known databases, such as GenBank of the National Center for Biotechnology Information (NCBI), however the source is not limited thereto.

The term "guide RNA" as used herein refers to a target gene or nucleic acid specific RNA that can direct a CRISPR enzyme to a target gene or target nucleic acid by binding to the CRISPR enzyme.

The guide RNA may consist of a crRNA comprising a sequence complementary to the gene or nucleic acid sequence to be targeted and a tracrRNA that binds the CRISPR enzyme, in which case the crRNA comprises a guide sequence that is a moiety capable of complementary binding to the gene or nucleic acid sequence to be targeted.

The leader sequence has a sequence complementary to the gene or nucleic acid sequence to be targeted, and functions to identify the gene or nucleic acid sequence to be targeted. In addition, the crRNA contains a portion whose sequence is complementary to a portion of the tracrRNA, and thus can partially complementarily bind to the tracrRNA.

In this case, the guide RNA may be a dual guide RNA in which crRNA and tracrRNA each independently exist; alternatively, the guide RNA may be a single guide RNA, wherein the crRNA and the tracrRNA are linked to each other, which may comprise a linker if the guide RNA is a single guide RNA. Depending on the type of CRISPR enzyme, the guide RNA may comprise only crRNA.

In another embodiment of the invention, the target-specific nuclease may be a Zinc Finger Nuclease (ZFN).

ZFNs include zinc finger proteins engineered to bind to a selected gene and a target site in a cleavage domain or cleavage half-domain. The zinc finger binding domain may be engineered to bind to a selected sequence. For example, reference may be made to Beerli et al (2002) Nature Biotechnol.20:135-141; pabo et al (2001) Ann. Rev. Biochem.70:313-340; isalan et al (2001) Nature Biotechnol.19:656-660; segal et al (2001) curr. Opin. Biotechnol.12:632-637; choo et al (2000) curr. Opin. Struct. Biol.10:411-416. Engineered zinc finger binding domains may have new binding specificities compared to naturally occurring zinc finger proteins. The engineering method includes rational design and various types of selection, but is not limited thereto. Rational design involves the use of databases containing, for example, triple (or quadruple) nucleotide sequences and single zinc finger amino acid sequences, in which case one or more zinc finger sequences that bind to a particular triple or quadruple sequence are combined with each triple or quadruple nucleotide sequence.

The selection of target sequences and the design and construction of fusion proteins (and polynucleotides encoding fusion proteins) are known to those skilled in the art, and are described in detail in U.S. patent publication Nos. 2005/0064474 and 2006/0188987, the entire contents of which are incorporated herein by reference.

Furthermore, as disclosed in these and other references, the zinc finger domains and/or multi-fingered zinc finger proteins can be linked together by any suitable linker sequence (e.g., a linker comprising more than five amino acids in length). For examples of linker sequences of more than six amino acids in length, reference may be made to U.S. Pat. nos. 6,479,626;6,903,185; and 7,153,949. The proteins described herein may comprise any combination of suitable linkers between the zinc fingers of the protein.

Furthermore, target-specific nucleases (e.g., ZFNs and/or meganucleases) can comprise a cleavage domain or cleavage half-domain.

It is well known that, for example, the cleavage domain may be heterologous to the DNA binding domain, due to the cleavage domain from a nuclease being different from the zinc finger DNA binding domain or the cleavage domain from a nuclease being different from the meganuclease DNA binding domain. The heterologous cleavage domain may be obtained from any endonuclease or exonuclease. Exemplary endonucleases that can be used as sources of cleavage domains include, but are not limited to, restriction endonucleases and meganucleases.

Restriction endonucleases (restriction enzymes) are present in a variety of species and can sequence-specifically bind to DNA (at a target site) thereby cleaving the DNA at or near the binding site. Some restriction enzymes (e.g., type IIS) cleave DNA at sites remote from the recognition site and have separable binding and cleavable domains. For example, the type IIS enzyme fokl catalyzes double-stranded cleavage of DNA at 9 nucleotides from the recognition site on one strand and 13 nucleotides from the recognition site on the other strand. Thus, in one embodiment, the fusion protein comprises at least one cleavage domain from a type IIS restriction enzyme and one or more zinc finger domains (which may or may not be engineered).

The term "TALE domain" as used herein refers to a protein domain that is capable of binding nucleotides in a sequence-specific manner by means of one or more TALE repeat modules. The TALE domain comprises at least one TALE repeat module, preferably from 1 to 30 TALE repeat modules, but is not limited thereto. In the present invention, the terms "TAL effector domain" and "TALE domain" are used interchangeably. The TALE domain may comprise half of the TALE repeat module.

Multiple target

The invention features the use of simultaneous target systems or multiple target systems capable of simultaneously recognizing on-target effects (activity) and/or off-target effects (activity).

The "target" of the present invention is the object to be cleaved or modified by a target-specific nuclease, and can be used interchangeably with the target or desired gene or desired nucleic acid or sequence thereof.

The portion of the "target" capable of recognizing the target-specific nuclease includes a nucleic acid sequence complementary to the target gene or nucleic acid sequence. The nucleic acid may be RNA, DNA, or RNA/DNA hybrids, but is not limited thereto.

The term "multiple targets" as used herein is a concept comprising one or more on-target effects and one or more off-target effects, and refers to a system formed that is capable of recognizing targets of on-target and off-target effects of a test nuclease simultaneously or individually.

Thus, a "multiplexed target" can be a gene or nucleic acid sequence targeted by a target-specific nuclease, a multiplexed target can be more than two targets, and a multiplexed target can consist of an in-target and an off-target.

By target-target is meant the sequence or position of a target gene or nucleic acid to which a target-specific nuclease complementarily binds; off-target refers to the sequence or location of a target gene or nucleic acid to which a target-specific nuclease moiety complementarily binds, at which undesirable nuclease activity occurs. Off-target targets can be sequences or locations of genes or nucleic acids that are not targeted by the target-specific nuclease, or can be nucleic acid sequences having less than 100% sequence homology to the nucleic acid sequence of the on-target. A nucleic acid sequence having less than 100% sequence homology with the nucleic acid sequence of the on-target is a nucleic acid sequence similar to the nucleic acid sequence of the on-target and may be a nucleic acid sequence comprising one or more different base sequences therein or having one or more base sequences deleted therein.

The multiplex targets may consist of one or more in-target targets and one or more off-target targets, each in unlimited number; in addition, the number of in-target and off-target targets can vary. That is, the multiplexed targets may be multiplexed targets comprising one in-target and two off-target targets; further, the multiplex target may be a multiplex target comprising two in-target targets and five off-target targets, and the design of the multiplex target may not be limited thereto.

In addition, in order to improve the selection reliability of the screening system of the present invention, objects to be cleaved or modified by a target-specific nuclease may be designed as a plurality of targets, desired genes, nucleic acids, or sequences thereof. The plurality of targets means that the number of targets is two or more, and the number of targets may be 2, 3, 4, 5, 6, 7 or more, and the number thereof is not limited.

In this case, the configuration of the system can be designed based on the multiplicity of targets and the multiple targets.

Multiple targets are linked to a selection element capable of recognizing on-target and off-target effects.

Selection element

The term "selection element" of the present invention refers to a label for detecting whether a target gene or nucleic acid sequence is cleaved or modified by a target-specific nuclease. Thus, a "selection element" may be used interchangeably with a "selection marker".

Based on whether the selection element is expressed, it can be detected whether the target gene or nucleic acid sequence is cleaved or modified by a target-specific nuclease. That is, it is possible to detect whether the in-target effect is increased and/or whether the off-target effect is decreased.

The selection element may be a toxin gene, an antibiotic resistance gene, a gene encoding a fluorescent protein, a tagging gene, a lacZ gene, or a gene encoding luciferase, but is not limited thereto.

A "toxin gene" is a gene or nucleic acid sequence that encodes a toxic substance (protein), the expression of which produces the toxic substance, and the viability (viability) of cells containing the toxin gene can be regulated by the toxic substance produced. Alternatively, the toxin gene may be a substance capable of regulating cell viability by affecting the synthesis of a substance required for viability of a cell containing the toxin gene or decomposing a substance required for viability of a cell containing the toxin gene.

The toxin gene may be Hok, fst, tisB, ldrD, flmA, ibs, txpA/BrnT, symE, XCV1262, ccdB, parE, mazF, yafO, hicA, kid, zeta, darT, sxt gene, hypoxanthine Phosphoribosyltransferase (HPRT) gene, or URA3, but is not limited thereto.

When the host is Escherichia coli (Escherichia coli), the toxin gene ccdB can be used.

When the host cell is a mammalian cell, the toxin gene HPRT may be used.

When the host is yeast, the toxin gene URA3 can be used.

An "antibiotic resistance gene" may be a gene or nucleic acid sequence that enables the expression of antibiotic resistance properties in an antibiotic-exposed state.

The antibiotic may be kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin (bleomycin), erythromycin, polymyxin B, tetracycline, zeocin, puromycin or chloramphenicol, but is not limited thereto.

The "fluorescent protein" is a protein exhibiting fluorescence, and may be, but not limited to, green Fluorescent Protein (GFP), red Fluorescent Protein (RFP), yellow Fluorescent Protein (YFP), cyan Fluorescent Protein (CFP), blue Fluorescent Protein (BFP), or Orange Fluorescent Protein (OFP).

The tag can be an AviTag, calmodulin tag, polyglutamic acid tag, E tag, FLAG tag, HA tag, his tag, myc tag, NE tag, S tag, SBP tag, softag1, softag 3, strep tag, TC tag, V5 tag, VSV tag, xpress tag, isopeptag, spyTag, snoeptag, biotin Carboxyl Carrier Protein (BCCP), glutathione-S-transferase (GST) tag, haloTag, maltose Binding Protein (MBP) tag, nus tag, thioredoxin tag, chitin Binding Protein (CBP), thioredoxin (TRX), or poly (NANP). In addition, the tag may use a fluorescent protein.

As a specific embodiment of the present invention, one or more selection elements can be used as selection elements for recognizing on-target and off-target effects of nucleases.

As an example, when using the lacZ gene as a selection element, if the target gene or nucleic acid sequence comprising lacZ is cleaved or modified by a target-specific nuclease, lacZ is not expressed, which can be detected by the blue-white screening method. The blue-white spot screening method is a screening method by means of beta-galactosidase expressed by lacZ gene, when colorless 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) is cleaved by beta-galactosidase, 5-bromo-4-chloro-indole is formed and spontaneously becomes dimer, thereby forming 5,5 '-dibromo-4,4' -dichloro-indigo, which is an insoluble pigment and oxidized to exhibit a vivid color. Therefore, whether the lacZ gene is expressed or not can be recognized by the phenomenon that cells expressing beta-galactosidase appear blue in the presence of X-gal, and cleavage or modification of a target gene or nucleic acid sequence comprising the lacZ gene can be predicted by the same means.

As another example, when a target-specific nuclease is screened by using a targeted target containing a toxin gene as a selection element and an off-target present in the genome of a cell, if a vector containing the targeted target and a vector containing the target-specific nuclease are introduced into the cell, it can be expected that the target-specific nuclease exhibiting high specificity cleaves the targeted target (with the result that expression of the toxin gene is suppressed) without cleaving the off-target (with the result that the genome is not cleaved). Thus, cells into which a target-specific nuclease with high specificity is introduced and in which only the target is cleaved but the off-target is not cleaved can survive; whereas cells in which both the on-target and off-target targets are cleaved do not survive because the cleavage of the off-target results in the genome being cleaved, thereby distinguishing the two types of cells from each other.

As yet another example, when a target-specific nuclease is screened by using an in-target comprising a toxin gene as a selection element and an off-target comprising a gene encoding a fluorescent protein as a selection element, if a vector comprising the in-target and a vector comprising the target-specific nuclease are introduced into a cell, it can be expected that the target-specific nuclease exhibiting high specificity cleaves the in-target (as a result of which expression of the toxin gene is inhibited) without cleaving the off-target (as a result of which the fluorescent protein is expressed). Therefore, cells into which a target-specific nuclease with high specificity is introduced and in which only a target of interest is cleaved and an off-target is not cleaved can survive, and can be distinguished using a fluorescent protein.

Thus, target-specific nucleases with high specificity that effectively cleave only the target-in-target can be selected based on whether these selection elements are expressed or not.

Selection of nucleases

By using the screening system, cells containing a target-specific nuclease having high specificity or high activity among the test nucleases are sorted, and a desired nuclease can be selected (identified) therefrom.

As a specific embodiment, the present invention provides a method of selecting for a target-specific nuclease, the method comprising:

a) Introducing component i) and component ii) of the screening system into the cell;

b) Sorting said cells by identifying target and off-target effects based on whether the selection element in a) is expressed; and

c) Selecting the nuclease present in the cells sorted in b) as a target-specific nuclease having high specificity or high activity.

For introducing the components i) and ii) into cells, publicly known methods can be used.

In some embodiments, the cells are transfected. Suitable transfection methods include: calcium phosphate mediated transfection, nuclear transfection, electroporation, cationic polymer transfection (e.g., DEAE-dextran or polyethyleneimine), viral transfection, virosome (virome) transfection, viral particle (viron) transfection, lipofection, cationic lipofection, immunoliposomal transfection, non-lipofection, dendrimer (dendrimer) transfection, heat shock transfection, magnetic transfection, lipofection, gene gun delivery, immunoperfection, optical transfection, and specific agonists that enhance nucleic acid uptake. Transfection methods are widely known in the art (see, e.g., "Current Protocols in Molecular Biology", ausubel et al, john Wiley & Sons, new York,2003 or "Molecular Cloning: A Laboratory Manual", sambrook & Russell, cold Spring Harbor Press, cold Spring Harbor, NY, third edition, 2001). In a preferred embodiment, component i) and component ii) can be introduced into the cell by electroporation.

In one embodiment, the DNA encoding component i) or component ii) may be present in a vector. Suitable vectors include plasmid vectors, phagemids, cosmids, artificial/kid chromosomes transposons and viral vectors (e.g., lentiviral vectors, adeno-associated viral vectors, etc.).

The term "vector" as used herein refers to a carrier nucleic acid molecule for transfer of a desired (intended, specific or required) nucleic acid; the vector may be a single-stranded nucleic acid, a double-stranded nucleic acid, or a partially double-stranded nucleic acid; in addition, the vector may comprise one or more free ends or no free ends (e.g., circular).

The vector may contain a "promoter" as a regulatory element for regulating the expression of the gene. The promoter may vary depending on the cell into which the vector is introduced. The promoter can be a polymerase III (pol III) promoter (e.g., U6 promoter, H1 promoter, etc.), a polymerase II (pol II) promoter (e.g., retroviral Rous Sarcoma Virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, beta-actin promoter, phosphoglycerate kinase (PGK) promoter, EF1 alpha promoter, etc.), or a polymerase I (pol I) promoter.

In another specific embodiment, the CRISPR-Cas system as component i) may be present in the form of a Ribonucleoprotein (RNP) wherein the guide RNA and the CRISPR enzyme form a complex.

Meanwhile, in b), the cells in which the on-target effect is increased and the off-target effect is decreased are sorted based on whether the selection element is expressed or not for recognition.

Further, in c), the nuclease obtained from the sorted cells is sequenced to obtain a sequence of the nuclease, thereby obtaining a target-specific nuclease having high specificity or high activity. The sequence of the target-specific nuclease obtained by this method can be used to prepare a nuclease for gene editing or the like.

As a method for obtaining the sequence of the obtained nuclease, for example, the following method can be performed.

That is, for example, when colonies were grown to a size that can be seen with the naked eye by culturing electroporated E.coli on LB agar plates containing kanamycin, chloramphenicol, and arabinose at 30 ℃ for 16 hours, each colony was inoculated into 10ml of LB medium containing chloramphenicol, followed by culture at 42 ℃ for 12 hours in a shaker. After centrifugation to obtain E.coli pellet, the plasmid was extracted from the pellet using a miniprep kit. The extracted plasmids can be sequenced using, for example, sanger sequencing methods. For the primers, universal sequencing primers (e.g., CMV-F or BGH-R) present outside the N-and C-termini of Cas9 can be used, as well as sequencing primers of the middle portion of Cas9.

Screening kit

Furthermore, the present invention provides a screening kit for selecting a target-specific nuclease having high specificity or high activity, the screening kit comprising: a multiplex target comprising one or more selection elements. The kit may be in the form of a composition.

In this case, the multiplex targets comprise one or more in-target targets and one or more off-target targets, which are described as above.

If necessary, the kit of the present invention can be provided as a liquid-1 type (one-type) kit comprising multiple targets comprising one or more selection elements.

If necessary, the kit of the present invention can be provided as a 2-liquid type (two liquid-type) kit comprising multiple targets and host cells containing one or more selection elements, respectively.

In this case, the kit can also be provided as a 1-liquid version by including multiple targets comprising selection elements in the host cell. For example, the kit may be contained in the host cell as a separate plasmid or may be provided by integration into the host cell genome.

If necessary, the kit of the present invention can be provided as a3 liquid type (three-type) kit comprising a multiplex target containing one or more selection elements, a host cell and a test nuclease, respectively.

In this case, the kit may also be provided as a 2-fluid version by including multiple targets comprising selection elements in the host cell. For example, the kit may be contained in the host cell as a separate plasmid or may be provided by integration into the host cell genome.

As described above, the present invention relates to a system for selecting a target-specific nuclease system having high specificity and high activity; preferably, the present invention encompasses both systems for screening and selecting target-specific nuclease systems by using a multiplex target system capable of recognizing off-target and on-target activities simultaneously, and various uses thereof.

The following detailed description describes exemplary embodiments using the principles of the present invention, and features and advantages of the present invention will be better understood by reference to the following detailed description. These and other embodiments are illustrated by or are apparent from and are included in the following detailed description.

Advantageous effects

There is still a problem that off-target occurs when a gene is corrected or manipulated by using a target-specific nuclease. To overcome this problem, it is important to develop target-specific nucleases with high specificity without the occurrence of off-target.

Therefore, the present invention has developed a system for screening target-specific nucleases having high specificity or high activity. The screening system of the present invention is a system capable of selecting a target-specific nuclease with reduced off-target effect and improved on-target effect, and by using multiple targets, it can be advantageously used for screening and selecting a target-specific nuclease with high specificity and/or high activity capable of satisfying both reduction of off-target effect and improvement of on-target effect.

In addition, when designing a multiplex target, the selection means included in the multiplex target can be variously designed according to the purpose of experiment, and can be used for selecting a target-specific nuclease by using various detection methods according to the selection means.

Drawings

Figure 1 schematically illustrates a screening method for CRISPR-Cas systems using multiple targets.

Plasmid a contains the toxic ccdB gene and the target site for NGG pam after the arabinose promoter. Plasmid B contains sgRNA under the control of the PltetO-1 promoter, which cleaves the target site of plasmid a in the presence of Cas9. The e.coli genomic DNA sequence is mismatched to the sgRNA sequence. Plasmid C contains the CMV-pltetO-1 dual promoter controlled Cas9 library sequence.

Fig. 2 is a diagram illustrating the results of screening according to the CRISPR-Cas9 system for designing off-target nucleic acid sequences in multiple targets.

The map is based on the use of the sequence EMX-1 inserted into the genomic DNA of E.coli and results of control experiments performed on plasmid B containing two mismatched sequences. CK means the number of colonies formed per unit on LB agar medium containing chloramphenicol and kanamycin, and CKA means the number of colonies formed per unit on LB agar medium containing chloramphenicol, kanamycin and arabinose. When two mismatches are located near the seed region (seed region) (56-WT-CAS 9), less than 0.1% of Cas 9-introduced E.coli survives. On the other hand, when an empty vector (instead of Cas 9) as a negative control was introduced into e.coli, about 0.01% of e.coli survived. The results of the negative control were considered as background due to the effect of plasmid A not functioning.

Fig. 3 is a diagram illustrating the results of screening according to the CRISPR-Cas9 system for designing off-target nucleic acid sequences in multiple targets.

This figure shows the results of a control experiment using the EMX-1 sequence inserted into the genomic DNA of E.coli and plasmid B containing a mismatch sequence. In the figure, CK means the number of colonies formed per unit on LB agar medium containing chloramphenicol and kanamycin, and CKA means the number of colonies formed per unit on LB agar medium containing chloramphenicol, kanamycin, and arabinose. When a mismatch is located near the seed region (7-WT-CAS 9), less than 0.1% of Cas 9-introduced E.coli survives. On the other hand, when an empty vector (instead of Cas 9) as a negative control was introduced into e.coli, about 0.01% of e.coli survived. The results of the negative control were considered as background due to the effect of plasmid A not functioning.

Fig. 4 is a diagram illustrating the results of screening Cas9 variants of a CRISPR-Cas9 system using multiple targets.

Plasmid A and plasmid B, a library of BW25141-EMX1 (7) containing 1 mismatch (7), and 3 types (mutant strains: XL-1Red competent cells, manufactured by Agilent Technologies, inc.. Agilent:Agilent (Agilent): agilent Technologies, inc. Genemorph II error-prone PCR kit, manufactured by Inc.. Titanium (Titanium): clontech, inc.) were screened. 1G1S (7) (1 generation 1 series): results of library screening of BW25141-EMX1 (7). 1G2S (7): the screening results of BW25141-EMX1 (7) screened by 1G1S (7). 1G3S (7): the screening results of BW25141-EMX1 (7) screened by 1G2S (7). 1G4S (17): the screening result of BW25141-EMX1 (17) screened by 1G3S (7). 1G 5S: (17): the screening result of BW25141-EMX1 (17) screened by 1G4S (17).

Fig. 5 is a diagram illustrating the results of screening Cas9 variants of a CRISPR-Cas9 system using multiple targets.

Plasmid A and plasmid B, a library of BW25141-EMX1 (56) containing 2 mismatches (56), and 3 types (mutant strains: derived from XL-1Red competent cells manufactured by Agilent Technologies, inc.. Agilent: agilent Technologies, inc. Genemorph II error-prone PCR kit manufactured by Titanium: clontech, inc.) were screened. 1G1S (1718) (1 generation 1 series): results of library screening of BW25141-EMX1 (1718). 1G2S (1718): the screening results of BW25141-EMX1 (1718) screened by 1G1S (1718) were obtained. 1G3S (1718): the screening results of BW25141-EMX1 (1718) screened by 1G2S (1718) were obtained. 2G1S (7): the screening result of BW25141-EMX1 (7) of 1G3S (17) shuffling library (shredded library). 2G2S (7): the screening results of BW25141-EMX1 (7) screened by 2G1S (7). 2G3S (7): the screening results of BW25141-EMX1 (7) screened by 2G2S (7). 2G4S (7): the screening result of 2G3S (7) screened BW25141-EMX1 (7). 2G5S (17): the screening result of BW25141-EMX1 (17) screened by 2G4S (7). 2G6S (17): the screening result of BW25141-EMX1 (17) screened by 2G5S (17).

FIGS. 6 and 7 are graphs illustrating the results of recognition of a reduced off-target activity of a Cas9 variant of a CRISPR-Cas9 system using multiple targets for specific genes (EMX 1 and T-DMD),

results illustrating the off-target activity and on-target activity of the DMD gene determined using a complete library (full-state library) obtained by screening performed by both methods simultaneously (fig. 6); and

results of off-target activity and on-target activity of the EMX1 gene measured using a complete library obtained by screening by performing both methods simultaneously are shown (FIG. 7).

FIGS. 8 and 9 are results obtained by performing on-target and off-target experiments on the DMD gene and the EMX1 gene, respectively, in mammalian cells using three clones selected by screening and results confirming improvement in specificity,

illustrates the on-target and off-target activity of three different clones (# 1, #20 and # 35) targeting human EMX-1 in HEK 293t cells (fig. 8); and

the on-target and off-target activity of three different clones (# 1, #20 and # 35) targeting human T-DMD in HEK 293T cells was demonstrated (FIG. 9).

Detailed Description

Definition of terms

The term "cleavage (cleavage, cleavage and/or cleavage)" refers to the act of generating a cleavage in a particular nucleic acid. As understood by those skilled in the art, cleavage (fragmentation) can leave blunt ends or sticky ends (i.e., 5 'or 3' overhanging ends). The term also includes single-stranded DNA cleavage ("nick") and double-stranded DNA cleavage.

The term "recombinant cell" refers to a host cell produced by genetically modifying a parent cell using genetic engineering techniques (i.e., recombinant techniques). The recombinant cell may comprise additions, deletions and/or modifications to the genomic nucleotide sequence of the parent cell.

The term "homology" refers to the identity between two or more nucleic acid sequences or two or more amino acid sequences. Sequence identity can be measured by% identity (or similarity or homology), with higher% being the higher the identity between sequences. Sequence identity is higher when homologs or orthologs of nucleic acid or amino acid sequences are aligned by using standard methods. For comparison, methods of sequence alignment are well known in the art. The following documents describe various programs and alignment algorithms [ references: smith and Waterman, adv.appl.math.2:482 1981; needleman and Wunsch, J.mol.biol.48:443 1970; pearson and Lipman, proc.natl.acad.sci.usa 85:2444 1988; higgins and Sharp, gene,73:237-44, 1988; higgins and Sharp, cabaos 5:151-3, 1989; corpet et al, nuc. 10881-90, 1988; huang et al, computer applications, biosc.8, 155-65, 1992; and Pearson et al, meth.mol.bio.24:307-31, 1994, altschul et al, j.mol.biol.215:403-10, 1990], and in particular methods of sequence alignment and homology calculation are described. For use with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx, documents are available from a variety of sources, including the internet and the national center for biological information (NCBI, national library of medicine, 38A, 8N805 room, bethesda, md.20894) [ references: NCBI Basic Local Alignment Search Tool (BLAST), altschul et al, J.mol.biol.215:403-10, 1990]. Further information can be found at the NCBI website.

The term "hybridization" refers to the reaction of one or more polynucleotides to form a complex that is stabilized by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonds may be formed by watson-crick base pairing, hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a double stranded structure, more than three strands forming a multi-stranded complex, a self-hybridizing single strand, or any combination thereof.

The term "expression" refers to the process of transcription of a polynucleotide from a DNA template (e.g., transcription into mRNA or another RNA transcript) and/or the process of translation of a transcribed mRNA into a peptide, polypeptide, or protein. The transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression in a eukaryotic cell may include splicing of the mRNA.

The term "selection element" or "selectable marker" refers to a gene that functions as a guide for the recognition and selection of on-target and/or off-target effects of the nucleases described herein. The selection element can include, but is not limited to, a toxin gene, a fluorescent marker, a luminescent marker, and a drug selection marker. Fluorescent labels can include, but are not limited to, genes encoding fluorescent proteins (e.g., green Fluorescent Protein (GFP), cyan Fluorescent Protein (CFP), yellow Fluorescent Protein (YFP), red fluorescent protein (dsRFP), etc.). Luminescent markers may include, but are not limited to, genes encoding luminescent proteins (e.g., luciferase). Drug selection markers suitable for use in the methods and compositions provided herein include, but are not limited to, antibiotic (e.g., ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, tetracycline, chloramphenicol, and neomycin) resistance genes. In some embodiments, the selection can be positive selection, that is, cells expressing the marker are isolated from the population, e.g., to produce an enriched population of cells comprising the selectable marker. In another embodiment, the selection can be a negative selection (negative selection), that is, the population is isolated from the cells, e.g., resulting in an enriched population of cells that does not contain the selectable marker. The separation may be performed by any convenient technique suitable for the selection marker used. For example, when using fluorescent labeling, cells can be isolated by fluorescence-activated cell sorting; and when cell surface markers are inserted therein, cells can be isolated from a heterogeneous population by means of affinity separation techniques (e.g., "panning" using an affinity agent attached to a solid substrate, magnetic separation, affinity chromatography) or other convenient techniques.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA.

The present invention will be described in detail below with the aid of exemplary embodiments using the principles of the invention.

Screening system

1. Screening system

The present invention relates to screening systems for selecting target-specific nucleases using multiplexed targets comprising one or more in-target targets and one or more off-target targets.

The term "screening system for selecting target-specific nucleases" refers to a concept that includes all of the following: kits and compositions for screening target-specific nucleases with high specificity and activity, methods of use thereof, and various intermediates derived in the process. The invention described as "system" in this specification may be construed as a composition or method to suit the aspect of the corresponding invention, as long as the invention is useful for screening for the desired target-specific nuclease.

Accordingly, one embodiment of the present invention is:

a screening composition for selecting target-specific nucleases having high specificity or high activity, the composition comprising:

1-1 target-specific nucleases: CRISPR-Cas system

A "target-specific nuclease" can be composed of a moiety capable of specifically recognizing a target and a moiety capable of cleaving or modifying the recognized target.

The portion capable of specifically recognizing the target may vary depending on the target gene or the nucleic acid sequence.

The moiety capable of cleaving or modifying the identified target may cleave or modify the identified target. In this case, the moiety capable of specifically recognizing the target and the moiety capable of cleaving or modifying the recognized target may each be present independently, or may be present in the form of a functional partition as a single structure.

The target-specific nuclease may be a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -CRISPR associated protein (Cas) system, a Zinc Finger Nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), fokl, an endonuclease, or a combination thereof, and may preferably be a CRISPR-Cas system, but is not limited thereto.

The CRISPR-Cas system may consist of guide RNA and CRISPR enzyme.

In this case, the guide RNA may be a moiety capable of specifically recognizing a target, and the CRISPR enzyme may be a moiety capable of cleaving or modifying the recognized target.

1-1-1 guide RNA

The guide RNA may comprise a nucleic acid sequence that complementarily binds to the gene or nucleic acid sequence to be targeted.

The guide RNA may consist of a crRNA comprising a sequence complementary to the gene or nucleic acid sequence to be targeted and a tracrRNA that binds to a CRISPR enzyme.

In this case, the crRNA contains a guide sequence, which is a moiety capable of complementary binding to the gene or nucleic acid sequence to be targeted. The guide sequence has a sequence complementary to the gene or nucleic acid sequence to be targeted, which can function to identify the gene or nucleic acid sequence to be targeted.

The size of the guide sequence may be 5bp-50bp, 5bp-40bp, 5bp-30bp or 5bp-20bp, but is not limited thereto. Preferably, the leader sequence may have a size of 5bp to 20 bp.

The nucleic acid sequence of the guide sequence may include, but is not limited to, a nucleic acid sequence that complementarily binds to the sequence or position of the gene or nucleic acid to be targeted at a ratio of 50% to 100%.

In addition, the crRNA contains a portion whose sequence is complementary to a portion of the tracrRNA, so that the crRNA can partially complementarily bind the tracrRNA.

The guide RNA may be a dual guide RNA in which the crRNA and tracrRNA each independently exist. Alternatively, the guide RNA may be a single guide RNA in which crRNA and tracrRNA are linked to each other. In this case, the single guide RNA may comprise a linker.

Furthermore, depending on the type of CRISPR enzyme, the guide RNA may comprise only crRNA.

The guide RNA may comprise a chemical modification. In this case, the chemical modification may include the following chemical modifications: wherein one or two or more of the nucleic acids comprised by the guide RNA have phosphorothioate linkage, locked Nucleic Acid (LNA), 2 '-O-methyl 3' -phosphorothioate (MS) or 2 '-O-methyl 3' -thioPACE (MSP) modifications therein.

The guide RNA may be a guide RNA truncated at a part of its 5' end.

The nucleic acid sequence of the guide RNA can be designed based on the target gene or the nucleic acid sequence.

The guide RNA may be contained in a vector, in which case the vector may contain a promoter suitable for directing RNA expression, such as the PltETO-1, araPBAD, rhampPAD or T7 promoter.

The guide RNA may be an artificially synthesized guide RNA.

1-1-2 CRISPR enzymes

The CRISPR enzyme can be a nucleic acid having a sequence encoding the CRISPR enzyme.

A nucleic acid having a sequence encoding a CRISPR enzyme can be comprised in a vector. In this case, the vector may comprise a promoter suitable for CRISPR enzyme expression, such as CMV or CAG.

CRISPR enzymes can be polypeptides or proteins.

CRISPR enzymes can be codon optimized to suit the subject to be introduced.

The CRISPR enzyme can be a type II CRISPR enzyme or a type V CRISPR enzyme.

The type II CRISPR enzyme may be Cas9.

The type V CRISPR enzyme may be a Cpf1 enzyme.

Cas9 may be streptococcus pyogenes Cas9 (SpCas 9), staphylococcus aureus (Staphylococcus aureus) Cas9 (SaCas 9), streptococcus thermophilus Cas9 (StCas 9), neisseria meningitidis Cas9 (NmCas 9), campylobacter jejuni Cas9 (CjCas 9), or orthologs thereof, but is not limited thereto. Preferably, cas9 may be SpCas9 or CjCas9.

Cas9 may be an active Cas9 or an inactive Cas9.

The inactive Cas9 may include a fully inactive Cas9 and a partially inactive Cas9 (e.g., nickase).

With respect to Cas9, one or two or more amino acids present in RuvC, HNH, REC, and/or PI domains may be mutated.

Cas9 may comprise mutations of one or two or more amino acids in the set of amino acids consisting of D10, E762, H840, N854, N863 and D986 among the amino acids of SpCas9, or in the set of amino acids in other Cas9 orthologs corresponding thereto.

Cas9 may comprise mutations of one or two or more amino acids in the set of amino acids consisting of R780, K810, K848, K855, and H982 in the amino acids of SpCas9 or in the set of amino acids in other Cas9 orthologs corresponding thereto.

Cas9 may comprise mutations of one or two or more amino acids in the group of amino acids consisting of G1104, S1109, L1111, D1135, S1136, G1218, N1317, R1335 and T1337 among the amino acids of SpCas9 or in the group of amino acids in other Cas9 orthologs corresponding thereto.

Cpf1 may be Francisella novicida Cpf1 (FnCpf 1), aminococcus Cpf1 (AsCpf 1), mao Luojun Cpf1 (LbCpf 1) or orthologs thereof, but is not limited thereto.

Cpf1 may be active Cpf1 or inactive Cpf1.

Non-reactive Cpf1 may include fully non-reactive Cpf1 and partially non-reactive Cpf1 (e.g.nickase).

For Cpf1, one or two or more amino acids present in RuvC, nuc, WED, REC and/or PI domains may be mutated.

Cpf1 may comprise amino acids consisting of D917, E1006 or D1255 in amino acids of FnCpf 1; d908, E993 or D1263 of amino acids of AsCpf 1; mutation of one or two or more amino acids of the group of amino acids consisting of D832, E925, D947 or D1180 of the amino acids of LbCpf1 or of the amino acids of other Cpf1 orthologues corresponding thereto.

CRISPR enzymes can recognize Protospacer Adjacent Motifs (PAMs) in genes or nucleic acid sequences.

PAM can vary depending on the source of the CRISPR enzyme.

The CRISPR enzyme may further comprise a functional domain. In this case, the CRISPR enzyme may be an active CRISPR enzyme or an inactive CRISPR enzyme.

The functional domain may be a Heterologous Functional Domain (HFD).

The functional domain may be one selected from the group consisting of: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity and molecular switching (e.g. light inducible).

The functional domain may be one selected from the group consisting of: methylases, demethylases, phosphatases, thymidine kinases, cysteine deaminases and cytidine deaminases.

In order to link the functional domain to the CRISPR enzyme, a linker may be further comprised between the CRISPR enzyme and the functional domain.

The linker may be (a) n, (G) n, GGGS, (GGS) n, (GGGGS) n, (EAAAK) n, SGGGS, GGSGGSGGS, sgsetpgtsetasatpes, XTEN, or (XP) n, in which case n may be 1, 2, 3, 4, 5, 6, 7, or more. However, the linker and n are not limited thereto.

The CRISPR enzyme may further comprise a Nuclear Localization Sequence (NLS).

Mutations of 1-1-3 CRISPR enzymes

The CRISPR enzyme can be an active CRISPR enzyme or an inactive CRISPR enzyme.

Inactive CRISPR enzymes can include fully inactive CRISPR enzymes and partially inactive CRISPR enzymes (e.g., nickases).

The CRISPR enzyme can be a mutant CRISPR enzyme. That is, the CRISPR enzyme can be a non-naturally occurring CRISPR enzyme.

In this case, the mutant CRISPR enzyme can be a naturally occurring CRISPR enzyme in which at least one or more amino acids of the CRISPR enzyme are mutated, the mutation can be a substitution, deletion, addition, etc. of an amino acid.

The mutant CRISPR enzyme can comprise a mutation of one or two or more amino acids that are present in the amino acids of the RuvC domain of the naturally occurring CRISPR enzyme.

The RuvC domain may be a RuvCI, ruvCII or RuvCIII domain.

The mutant CRISPR enzyme can comprise a mutation of one or two or more amino acids present in the amino acids of the HNH domain of the naturally occurring CRISPR enzyme.

The mutant CRISPR enzyme can comprise a mutation of one or two or more amino acids present in the amino acids of the REC domain of the naturally occurring CRISPR enzyme.

The mutant CRISPR enzyme can comprise a mutation of one or two or more of the amino acids present in the PI domain of the naturally occurring CRISPR enzyme.

The mutant CRISPR enzyme can comprise a mutation of one or two or more amino acids present in the amino acids of the Nuc domain of the naturally occurring CRISPR enzyme.

The mutant CRISPR enzyme can comprise a mutation of one or two or more of the amino acids present in the WED domain of the naturally occurring CRISPR enzyme.

Further, the CRISPR enzyme may be a CRISPR enzyme that is recombinantly obtained by combining a portion of each mutant CRISPR enzyme.

For example, the RuvC domain, the HNH domain, and the PI domain of a CRISPR enzyme (i.e., a wild-type CRISPR enzyme) that is present in its native state can be replaced or recombined with the RuvC domain of CRISPR enzyme variant 1, the HNH domain of CRISPR enzyme variant 2, and the PI domain of CRISPR enzyme variant 3, respectively. Alternatively, new CRISPR variants other than

CRISPR enzyme variants

1, 2, 3 can be constructed by combining amino acids 1-500 of the amino acid sequence of CRISPR enzyme variant 1 (or a nucleic acid sequence encoding the same) with amino acids 501-1000 of the amino acid sequence of CRISPR enzyme variant 2 (or a nucleic acid sequence encoding the same) and amino acids 1001 through the end of the amino acid sequence of CRISPR enzyme variant 3 (or a nucleic acid sequence encoding the same).

As another example, a portion of CRISPR enzyme variant 1, e.g., amino acids 50-700 of its amino acid sequence (or its encoding nucleic acid sequence), can be replaced or recombined with amino acids 50-700 of the amino acid sequence of CRISPR enzyme variant 2 (or its encoding nucleic acid sequence).

Further, the CRISPR enzyme can be a CRISPR enzyme produced from a combination of different CRISPR enzymes.

For example, ruvC domain of Cas9 can be replaced or recombined with RuvC domain of Cpf1.

Furthermore, the CRISPR enzyme may be a CRISPR enzyme variant further comprising a functional domain in an active or inactive CRISPR enzyme.

The functional domain may be a Heterologous Functional Domain (HFD).

For example, a CRISPR enzyme variant may comprise a transcription repression domain in an inactive enzyme, which may be a variant that represses expression of a target gene or nucleic acid sequence. In addition, a CRISPR enzyme variant may comprise a cytidine deaminase in an inactive enzyme, which may be a variant capable of inhibiting or increasing the expression of a corresponding gene or nucleic acid sequence by altering a particular base sequence of the target gene or nucleic acid sequence. Mutational Effect of 1-1-4CRISPR enzymes

A mutant CRISPR enzyme can be a CRISPR enzyme that has been mutated such that the cleavage effect (i.e., on-target) of a gene or nucleic acid to be targeted is increased.

A mutant CRISPR enzyme can be a CRISPR enzyme that has been mutated such that the effect of cleavage (i.e., on-target) of a gene or nucleic acid to be targeted is reduced.

A mutant CRISPR enzyme can be a CRISPR enzyme that has been mutated such that the effect of cleavage (i.e., off-target) of a non-targeted gene or nucleic acid is increased.

A mutant CRISPR enzyme can be a CRISPR enzyme that has been mutated such that the effect of cleavage (i.e., off-target) of a non-targeted gene or nucleic acid is reduced.

The modified CRISPR enzyme can be a CRISPR enzyme modified such that the binding capacity to a gene or nucleic acid is increased.

The modified CRISPR enzyme can be a CRISPR enzyme modified such that the binding capacity to a gene or nucleic acid is reduced.

The modified CRISPR enzyme can be a CRISPR enzyme modified such that recognition of a Protospacer Adjacent Motif (PAM) in a gene or nucleic acid sequence is increased.

The modified CRISPR enzyme may be a CRISPR enzyme modified such that the ability to recognize PAM in a gene or nucleic acid sequence is reduced.

The modified CRISPR enzyme may be a CRISPR enzyme in which helicase kinetics are modified.

The modified CRISPR enzyme may comprise a conformational rearrangement depending on the modification.

1-2 CRISPR complexes

The CRISPR enzyme and guide RNA can form a CRISPR complex.

CRISPR complexes can be formed extracellularly.

CRISPR complexes can form in the cytoplasm of cells.

CRISPR complexes can be in cells is formed in the cell nucleus of (1).

In CRISPR complexes, CRISPR enzymes can recognize PAM present in the gene or nucleic acid sequence to be targeted.

In CRISPR complexes, guide RNAs can complementarily bind to a gene or nucleic acid sequence to be targeted.

When the CRISPR complex binds to a gene or nucleic acid sequence to be targeted, the CRISPR enzyme of the CRISPR complex can cut or modify the gene or nucleic acid sequence to be targeted.

In this case, a target-specific nuclease may be introduced into the cell.

The target-specific nuclease can be introduced into cells by transfection, microinjection, electroporation, or the like using a viral vector system, ribonucleoprotein (RNP), nanoparticles, liposomes, or the like, but the introduction method is not limited thereto.

The cell may be a prokaryotic cell or a eukaryotic cell.

1-3 multiple targets

The target may be a gene or nucleic acid sequence targeted by a target-specific nuclease.

The multiplex target may consist of a gene or nucleic acid sequence to be targeted (i.e., a medium target) as well as a non-targeted gene or nucleic acid sequence (i.e., a miss target).

The multiplexed targets may consist of one or more in-target targets and one or more off-target targets. As an embodiment, the multiplex target may consist of one in-target and one or more off-target targets. As another embodiment, the multiplex target may consist of one in-target and one off-target.

Multiple targets may be present in the cell or introduced from an external source.

When multiple targets are present in a cell, the multiple targets may be present in the genome of the host cell. Preferably, the one or more off-target targets may be present in the genome of the host cell.

When multiple targets are introduced into a cell from an exogenous source, multiple targets can be introduced using a vector system.

When multiple targets are introduced into a cell from an exogenous source, a vector system can be used to introduce the multiple targets.

When multiple targets are introduced into a cell from an external source, multiple targets can be introduced by using different vectors for each target. That is, when the multiplex target consists of one on-target and one off-target, the vector may consist of a vector containing the on-target and a vector containing the off-target.

Multiple targets can be introduced into cells by transfection, microinjection, electroporation, or the like using a viral vector system, nanoparticles, liposomes, or the like, but the introduction method is not limited thereto.

1-3-1 targeting

The target-in-target can be the sequence or location of a target gene or nucleic acid to which the target-specific nuclease complementarily binds.

The target-in-target can be a sequence or location of a gene or nucleic acid having sequence complementarity to a nucleic acid sequence of a portion of the target-specific nuclease capable of specifically recognizing the target.

When the target-specific nuclease is a CRISPR-Cas system, the target-in-target can be a sequence or location of a gene or nucleic acid that complementarily binds to a guide RNA or a guide sequence of a guide RNA.

In this case, the sequence of the gene or nucleic acid that complementarily binds to the guide RNA or the guide sequence of the guide RNA may be 5 to 50bp; in addition, the length of the guide RNA or the guide sequence of the guide RNA can be adjusted according to the length of the nucleic acid sequence.

When the target-specific nuclease is a CRISPR-Cas system, the mid-target can comprise a PAM sequence that is recognized by the CRISPR enzyme.

When the target-specific nuclease is a CRISPR-Cas system, the target-in-target can comprise a nucleic acid sequence that complementarily binds to a guide RNA or a guide sequence of a guide RNA and a PAM sequence that is recognized by the CRISPR enzyme.

In this case, the nucleic acid sequence in the target-in-target that complementarily binds to the guide RNA or the guide sequence of the guide RNA may be located at the 5' end of the PAM sequence recognized by the CRISPR enzyme.

In this case, the nucleic acid sequence in the target-in-target that complementarily binds to the guide RNA or the guide sequence of the guide RNA may be located at the 3' end of the PAM sequence recognized by the CRISPR enzyme.

The target of the target can be (N) _5-50 PAM or PAM- (N) _5-50 (ii) a In this case, N may be A, T, G or C; or A, U, G or C.

In addition, the on-target may be located in the genome of the cell.

The cell may be a prokaryotic cell or a eukaryotic cell.

In this case, the on-target may comprise a selection element.

The selection element may be a toxin gene, an antibiotic resistance gene, a gene encoding a fluorescent protein, a tag gene, a lacZ gene, or a gene encoding luciferase, but is not limited thereto.

The toxin gene may be Hok, fst, tisB, ldrD, flmA, ibs, txpA/BrnT, symE, XCV1262, ccdB, parE, mazF, yafO, hicA, kid, zeta, darT or Sxt gene, and preferably may be CcdB, but is not limited thereto.

The toxin gene may vary depending on the source of the targeted target (i.e., depending on the origin). That is, those skilled in the art can appropriately select and use a toxin gene depending on the type of host cell.

When the host is Escherichia coli, the toxin gene ccdB can be used.

When the host cell is a mammalian cell, the toxin gene Hypoxanthine Phosphoribosyltransferase (HPRT) may be used.

When the host is yeast, the toxin gene URA3 can be used.

The antibiotic resistance gene can be designed in a diversified manner according to the antibiotic type.

The antibiotic may be kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, zeocin, puromycin or chloramphenicol, but is not limited thereto.

The tag can be an AviTag, calmodulin tag, polyglutamic acid tag, E tag, FLAG tag, HA tag, his tag, myc tag, NE tag, S tag, SBP tag, softag1, softag 3, strep tag, TC tag, V5 tag, VSV tag, xpress tag, isopeptag, spyTag, snoeptag, biotin Carboxyl Carrier Protein (BCCP), glutathione-S-transferase (GST) tag, haloTag, maltose Binding Protein (MBP) tag, nus tag, thioredoxin tag, chitin Binding Protein (CBP), thioredoxin (TRX), or poly (NANP).

In this case, the selection element may not be expressed when the target-in-target comprising the selection element is cleaved or modified by the target-specific nuclease.

1-3-2 off-target targets

Off-target targets can be sequences or locations of non-target genes or nucleic acids to which target-specific nuclease moieties complementarily bind.

The off-target may be a nucleic acid sequence comprising one or more other base sequences in the on-target.

Off-target targets can be a sequence or location of a gene or nucleic acid having a sequence that is complementary to a portion of the nucleic acid sequence in the target-specific nuclease capable of specifically recognizing a portion of the target.

When the target-specific nuclease is a CRISPR-Cas system, the off-target can be a sequence or position of a gene or nucleic acid that is partially complementary bound to a guide RNA or a guide sequence of a guide RNA.

When the target-specific nuclease is a CRISPR-Cas system, the off-target may comprise a PAM sequence that is recognized by the CRISPR enzyme.

When the target-specific nuclease is a CRISPR-Cas system, the off-target can comprise a nucleic acid sequence that complementarily binds to a guide RNA or a guide sequence of a guide RNA and a PAM sequence that is recognized by the CRISPR enzyme.

In this case, the nucleic acid sequence in the off-target that complementarily binds to the guide RNA or the guide sequence of the guide RNA may be located at the 5' end of the PAM sequence recognized by the CRISPR enzyme.

In this case, the nucleic acid sequence in the off-target that complementarily binds to the guide RNA or the guide sequence of the guide RNA may be located at the 3' end of the PAM sequence recognized by the CRISPR enzyme.

The miss target may be (N) _5-50 PAM or PAM- (N) _5-50 (ii) a In this case, N may be A, T, G or C; or A, U, G or C.

Off-target targets can be sequences or locations of genes or nucleic acids that comprise non-complementary binding to one or more of the nucleic acid sequences of the portions of the target-specific nucleases that specifically recognize the target.

Off-target targets can be sequences or locations comprising bound genes or nucleic acids that are less than 100% complementary to the nucleic acid sequence of the portion of the target-specific nuclease that specifically recognizes the target. A nucleic acid sequence having less than 100% sequence homology is a nucleic acid sequence that is similar to a nucleic acid sequence of a target, and may be a nucleic acid sequence that comprises one or more different base sequences therein or in which one or more base sequences are deleted.

When the target-specific nuclease is a CRISPR-Cas system, the off-target can be a sequence or location of a gene or nucleic acid comprising one or more non-complementary binding to a guide RNA or a nucleic acid sequence of a guide RNA.

When the target-specific nuclease is a CRISPR-Cas system, the off-target can be a sequence or location of a gene or nucleic acid comprising binding less than 100% complementary to the nucleic acid sequence of the guide RNA or the guide sequence of the guide RNA.

When the target-specific nuclease is a CRISPR-Cas system, the off-target can comprise one or more mismatched nucleic acid sequences in a PAM sequence recognized by the CRISPR enzyme.

In this case, the off-target may be located in the genome of the cell.

The cell may be a prokaryotic cell or a eukaryotic cell.

In this case, the off-target may comprise a selection element.

In this case, the selection element may not be expressed when the off-target comprising the selection element is cleaved or modified by the target-specific nuclease.

In this case, the selection element may be expressed when the off-target comprising the selection element is not cleaved or modified by the target-specific nuclease.

1-4 Effect of selection of elements

The selection element comprised in the on-target and/or off-target targets may be an element that determines the high specificity or activity of the target-specific nuclease.

As an example, when a target-specific nuclease is screened by using an in-target containing a toxin gene as a selection element and an off-target containing an antibiotic resistance gene as a selection element, if a vector each containing the in-target and the off-target and a vector containing the target-specific nuclease are introduced into a cell, it can be expected that a target-specific nuclease exhibiting high specificity cleaves the in-target (with the result that expression of the toxin gene is suppressed) without cleaving the off-target (with the result that the antibiotic resistance gene is expressed). Therefore, cells into which a target-specific nuclease with high specificity is introduced and in which only the target is cleaved but the off-target is not cleaved can survive treatment with an antibiotic. Therefore, the temperature of the molten metal is controlled, target-specific nucleases with high specificity or activity can be screened based on whether these selection elements are expressed.

As another example, when a target-specific nuclease is screened by using an in-target containing a toxin gene as a selection element and an off-target present in the genome of a cell, if a vector containing the in-target and a vector containing the target-specific nuclease are introduced into the cell, it can be expected that the target-specific nuclease exhibiting high specificity cleaves the in-target (with the result that expression of the toxin gene is suppressed) without cleaving the off-target (with the result that the genome is not cleaved). Thus, cells into which a target-specific nuclease with high specificity is introduced and in which only the target is cleaved but the off-target is not cleaved can survive; whereas cells in which both the on-target and off-target targets are cleaved do not survive because the cleavage of the off-target results in the genome being cleaved, thereby distinguishing the two types of cells from each other.

As yet another example, when a target-specific nuclease is screened by using an in-target comprising a toxin gene as a selection element and an off-target comprising a gene encoding a fluorescent protein as a selection element, if a vector comprising the in-target and a vector comprising the target-specific nuclease are introduced into a cell, it can be expected that the target-specific nuclease exhibiting high specificity cleaves the in-target (as a result of which expression of the toxin gene is inhibited) without cleaving the off-target (as a result of which the fluorescent protein is expressed). Therefore, cells into which a target-specific nuclease with high specificity is introduced and in which only the target is cleaved but the off-target is not cleaved can survive, and can be distinguished using a fluorescent protein.

As described above, the method for screening target-specific nucleases can be variously designed according to the selection element.

2. Screening method

Target-specific nucleases with high specificity or activity can be screened using a screening system.

As one aspect, the invention relates to a method of screening for target-specific nucleases having high specificity or high activity in a plurality of target-specific nuclease test sets.

In the present invention, the term "method of screening for a target-specific nuclease/method of screening for a target-specific nuclease" refers to sorting cells containing a target-specific nuclease having high specificity or high activity. That is, the method refers to a step prior to a step of recognizing a specific nuclease.

In particular, the method is very useful in selecting a target-specific nuclease having high specificity or high activity from a variety of nuclease variants prepared by improving target-specific nucleases and nuclease libraries.

2-1 first embodiment of screening method

As a specific embodiment of the method of the present invention,

the method is useful as a method for screening target-specific nucleases with high specificity and activity from a variety of target-specific nuclease "variants".

The method is a method using the following components:

i) A multiplex target comprising one or more in-target targets and one or more off-target targets; and

ii) a target-specific nuclease variant,

the method comprises the following steps:

a) Introducing component i) and component ii) into a cell; and

b) Sorting cells in a) that are only modified for the target.

2-1-1 component

In this case, component i) can use a vector system, and in component i), the in-target and off-target targets can be contained in separate vectors.

The target-specific nuclease of component ii) may be a CRISPR-Cas system and may be a ZFN, TALEN, fokI or a mixture thereof, and furthermore, is not limited thereto.

The CRISPR-Cas system may consist of a guide RNA and a CRISPR enzyme.

The target-specific nuclease variant of component ii) may be a CRISPR-Cas system variant, preferably may be a CRISPR enzyme variant, but is not limited thereto. Nuclease variants can be produced using electromagnetic waves, UV, irradiation, chemicals, exogenous/endogenous gene action, and the like. In one example, the variants are obtained by irradiating the WT CRISPR-Cas system with UV.

The CRISPR enzyme can be Cas9 and the CRISPR enzyme variant can be a Cas9 variant.

The Cas9 variant may be a variant in which the on-target effect is increased or decreased. In this case, an on-target effect means that the on-target position is cleaved or modified by Cas9.

The Cas9 variant may be a variant in which off-target effects are increased or decreased. In this case, off-target effect means that the off-target position is cleaved or modified by Cas9.

Preferably, the Cas9 variant may be a variant in which the on-target effect is increased and/or the off-target effect is decreased.

Component ii) may use a vector system, and when component ii) is a CRISPR-Cas system, the guide RNA and CRISPR enzyme may be comprised in the same vector or in different vectors.

Component ii) may use a Ribonucleoprotein (RNP) system, and when component ii) is a CRISPR-Cas system, the guide RNA and CRISPR enzyme may be in the form of an RNA-protein complex.

Component ii) may be artificially synthesized.

When component ii) is a CRISPR-Cas system, the guide RNA may be artificially synthesized, and the CRISPR enzyme may also be an artificially synthesized protein or polypeptide.

In component i), the target-in-place can be a sequence or location of a gene or nucleic acid that complementarily binds to the guide RNA or a guide sequence of the guide RNA of component ii).

In component i), the off-target may be a sequence or position of a gene or nucleic acid that is partially complementary to the guide RNA or guide sequence of the guide RNA of component ii).

In component i), the off-target may be a sequence or position of a gene or nucleic acid that forms one or more non-complementary binding to the guide RNA of component ii) or the nucleic acid sequence of the guide RNA.

In component i), the on-target and/or off-target targets may comprise a selection element.

2-1-2 Process

In this case, when the components i) and ii) are introduced into the cells in a),

the components i) and ii) may be introduced into cells by transfection, microinjection, electroporation, etc., using a viral vector system, ribonucleoprotein (RNP), nanoparticles, liposomes, etc., but the introduction method is not limited thereto.

The cell may be a prokaryotic cell or a eukaryotic cell.

In addition, off-target targets may be present in the genome of the cell.

When the off-target in component i) is present in the genome of the cell, the vector comprising the on-target of component i) and component ii) can be introduced into the cell.

In addition, the on-target may be present in the genome of the cell.

When the on-target in component i) is present in the genome of the cell, a vector comprising the off-target of component i) and component ii) can be introduced into the cell.

In the step (b) of the above-mentioned process,

among the cells into which components i) and ii) have been introduced in a), cells in which only the targeted targets in component i) are cleaved by component ii) can be sorted out.

Cells in which only the on-target is cleaved can be selected by inhibiting the expression of the selection element contained in the on-target.

For example, when the intermediate target comprises a toxin gene, the cell can survive because the intermediate target is cleaved such that expression of the toxin gene is inhibited.

In addition, cells in component i) that are not cleaved by component ii) for off-target targets can be sorted.

Cells in which the off-target is not cleaved can be selected by expressing the selection element because the off-target is not cleaved.

For example, when the off-target comprises an antibiotic resistance gene, cells that are antibiotic resistant can be sorted in the presence of an antibiotic by allowing the antibiotic resistance gene to be expressed because the off-target is not cleaved.

Alternatively, when the off-target is located in the genome of the cell, the genome of the cell is normally retained since it is not cleaved by component ii), with the result that the cell is able to survive.

In this case, the cells sorted in b) may be cells in which the on-target of component i) is cleaved by component ii) and the off-target of component i) is not cleaved by component ii). These cells can be distinguished by means of a selection element.

Thus, target-specific nucleases with high specificity and high activity that effectively cleave only a target of interest can be screened among a variety of target-specific nuclease variants by using the screening method described above, in which case the screening method can be designed with a variety of detection methods using a variety of selection elements.

2-2 second embodiment of the screening method

As a further specific embodiment, it is possible to,

the method is useful as a method for screening a "library" of target-specific nucleases for target-specific nucleases with high specificity and activity.

The method is a screening method using the following components:

ii) a library of target-specific nucleases,

the method comprises the following steps:

a) Introducing component i) and component ii) into a cell; and

b) Sorting cells in a) that are only modified for the target.

2-2-1 component

In this case, component i) may use a vector system, and in component i), the on-target and off-target targets may be contained in separate vectors.

The target-specific nuclease library can be a CRISPR-Cas system library. Nuclease libraries can be prepared directly, can be commercially available, and can be obtained from databases.

The CRISPR-Cas system may consist of guide RNA and CRISPR enzyme.

The CRISPR-Cas system library can be a guide RNA library and/or a CRISPR enzyme library.

Component ii) may use a Ribonucleoprotein (RNP) system and when component ii) is a CRISPR-Cas system, the guide RNA and CRISPR enzyme may be in the form of an RNA-protein complex.

Component ii) may be artificially synthesized.

When component ii) is a CRISPR-Cas system, the guide RNA may be artificially synthesized, CRISPR enzymes can also be artificially synthesized proteins or polypeptides.

In component i), off-target targets may be sequences or positions of genes or nucleic acids that form non-complementary binding to one or more of the nucleic acid sequences of the guide RNA or guide sequences of the guide RNA of component ii).

2-2-2 Process

The cell may be a prokaryotic cell or a eukaryotic cell.

In addition, off-target targets may be present in the genome of the cell.

In addition, the on-target may be present in the genome of the cell.

In the step (b) of the above-mentioned process,

Thus, target-specific nucleases with high specificity or high activity can be screened in a target-specific nuclease library via selected cells using the screening method as described above, in which case the screening method can be designed with a variety of detection methods using a variety of selection elements. Selection system

3. Selection system for target-specific nucleases

By using a screening system to select cells containing a target-specific nuclease having high specificity or activity from among the test nucleases, desired nucleases can be selected (identified) therefrom.

Another aspect of the invention relates to a selection system for target-specific nucleases using multiple targets comprising one or more on-target targets and one or more off-target targets.

The term "selection system for target-specific nucleases" refers to a concept that includes all of the following: kits and compositions for selecting target-specific nucleases with high specificity and activity and recognizing their specific composition, methods of use thereof and various intermediates derived in the process. The invention described as "system" in this specification may be construed as a composition or method to suit the aspect of the respective invention, as long as the invention is used to select the desired target-specific nuclease.

The selection system may consist of multiple targets and target-specific nucleases.

3-1 target-specific nucleases (CRISPR-Cas systems)

In this case, the target-specific nuclease may consist of a moiety capable of specifically recognizing the target and a moiety capable of cleaving or modifying the recognized target.

The portion capable of specifically recognizing the target may vary depending on the target gene or nucleic acid sequence.

The CRISPR-Cas system may consist of guide RNA and CRISPR enzyme.

In this case, the guide RNA may be a portion capable of specifically recognizing the target, and the CRISPR enzyme may be a portion capable of cleaving or modifying the recognized target.

3-1-1 guide RNA

In addition, the crRNA contains a portion whose sequence is complementary to a portion of the tracrRNA, and thus the crRNA may partially complementarily bind the tracrRNA.

The guide RNA may be a guide RNA truncated at a part of its 5' end.

The guide RNA may be an artificially synthesized guide RNA.

3-1-2 CRISPR enzymes

CRISPR enzymes can be polypeptides or proteins.

CRISPR enzymes can be codon optimized to suit the subject to be introduced.

The CRISPR enzyme may be a type II CRISPR enzyme or a type V CRISPR enzyme.

The type II CRISPR enzyme may be Cas9.

The type V CRISPR enzyme may be a Cpf1 enzyme.

Cas9 may be an active Cas9 or an inactive Cas9.

Cpf1 may be active Cpf1 or inactive Cpf1.

CRISPR enzymes can recognize Protospacer Adjacent Motifs (PAM) in genes or nucleic acid sequences.

PAM can vary depending on the source of the CRISPR enzyme.

The functional domain may be a Heterologous Functional Domain (HFD).

The CRISPR enzyme may further comprise a Nuclear Localization Sequence (NLS).

Modification of 3-1-3 CRISPR enzymes

The CRISPR enzyme may be an active CRISPR enzyme or an inactive CRISPR enzyme.

The RuvC domain may be a RuvCI, ruvCII or RuvCIII domain.

For example, the RuvC domain, HNH domain, and PI domain of a CRISPR enzyme (i.e., a wild-type CRISPR enzyme) that is present in nature can be replaced or recombined with the RuvC domain of CRISPR enzyme variant 1, the HNH domain of CRISPR enzyme variant 2, and the PI domain of CRISPR enzyme variant 3, respectively. Alternatively, new CRISPR variants other than

CRISPR enzyme variants

1, 2, 3 can be constructed by combining amino acids 1-500 (or encoding nucleic acid sequences thereof) of the amino acid sequence of CRISPR enzyme variant 1 with amino acids 501-1000 (or encoding nucleic acid sequences thereof) of the amino acid sequence of CRISPR enzyme variant 2 and amino acids 1001 to the terminus of the amino acid sequence of CRISPR enzyme variant 3 (or encoding nucleic acid sequences thereof).

Furthermore, the CRISPR enzyme may be a CRISPR enzyme variant further comprising a functional domain in an active CRISPR enzyme or an inactive CRISPR enzyme.

The functional domain may be a Heterologous Functional Domain (HFD).

For example, a CRISPR enzyme variant may comprise a transcription repression domain in an inactive enzyme, which may be a variant that represses expression of a target gene or nucleic acid sequence. In addition, a CRISPR enzyme variant may comprise a cytidine deaminase in an inactive enzyme, which may be a variant capable of inhibiting or increasing the expression of a corresponding gene or nucleic acid sequence by altering a particular base sequence of the target gene or nucleic acid sequence. Mutational Effect of 3-1-4CRISPR enzymes

A mutant CRISPR enzyme can be a CRISPR enzyme that has been mutated such that the effect of cleavage (i.e., on-target) of a gene or nucleic acid to be targeted is increased.

The modified CRISPR enzyme can be a CRISPR enzyme modified such that the ability to recognize PAM in a gene or nucleic acid sequence is reduced.

3-2 CRISPR complexes

The CRISPR enzyme and guide RNA can form a CRISPR complex.

CRISPR complexes can be formed extracellularly.

CRISPR complexes can form in the cytoplasm of cells.

CRISPR complexes can form in the nucleus of cells.

In this case, a target-specific nuclease may be introduced into the cell.

The cell may be a prokaryotic cell or a eukaryotic cell.

3-3 multiple targets

In this case, the target may be a gene or nucleic acid sequence targeted by a target-specific nuclease.

The multiplexed targets may consist of one or more on-target targets and one or more off-target targets. As one embodiment, the multiplex target may consist of one in-target and one or more off-target targets. As another embodiment, the multiplex target may consist of one on-target and one off-target.

3-3-1 target

The on-target can be a sequence or location of a target gene or nucleic acid to which the target-specific nuclease complementarily binds.

When the target-specific nuclease is a CRISPR-Cas system, the in-target can be a sequence or location of a gene or nucleic acid that is complementary to a guide RNA or a guide sequence of a guide RNA.

In this case, it is preferable that, the sequence of the gene or nucleic acid that complementarily binds to the guide RNA or the guide sequence of the guide RNA may be 5-50bp; in addition, the length of the guide RNA or the guide sequence of the guide RNA can be adjusted according to the length of the nucleic acid sequence.

When the target-specific nuclease is a CRISPR-Cas system, the target-in-target may comprise a PAM sequence that is recognized by the CRISPR enzyme.

In this case, the intermediate target may be located in the genome of the cell.

The cell may be a prokaryotic cell or a eukaryotic cell.

In this case, the on-target may comprise a selection element.

The toxin gene can be, but is not limited to, hok, fst, tisB, ldrD, flmA, ibs, txpA/BrnT, symE, XCV1262, ccdB, parE, mazF, yafO, hicA, kid, zeta, darT, or Sxt genes.

Toxin genes may vary depending on the source (i.e., according to origin) of the targeted target. That is, those skilled in the art can appropriately select and use a toxin gene depending on the type of host cell.

When the host is Escherichia coli, the toxin gene ccdB can be used.

When the host is yeast, the toxin gene URA3 can be used.

The fluorescent protein may be Green Fluorescent Protein (GFP), red Fluorescent Protein (RFP), yellow Fluorescent Protein (YFP), cyan Fluorescent Protein (CFP), blue Fluorescent Protein (BFP), or Orange Fluorescent Protein (OFP), but is not limited thereto.

In this case, the selection element may not be expressed when the in-target comprising the selection element is cleaved or modified by the target-specific nuclease.

3-3-2 off-target targets

Off-target targets can be nucleic acid sequences that include one or more additional base sequences in the target.

The off-target can be (N) _5-50 PAM or PAM- (N) _5-50 (ii) a In this case, N may be A, T, G or C; or A, U, G or C.

In this case, the off-target may be located in the genome of the cell.

The cell may be a prokaryotic cell or a eukaryotic cell.

In this case, the off-target may comprise a selection element.

3-4 Effect of selection elements

Based on whether the selection element is expressed, it can be detected whether the target gene or nucleic acid sequence is cleaved or modified by a target-specific nuclease. That is, it can be detected whether the on-target effect is increased and/or the off-target effect is decreased.

As an example, when a target-specific nuclease is screened by using a mid-target comprising a toxin gene as a selection element and an off-target comprising an antibiotic resistance gene as a selection element, if a vector each comprising the mid-target and the off-target and a vector comprising the target-specific nuclease are introduced into a cell, it can be expected that a target-specific nuclease exhibiting high specificity cleaves the mid-target (with the result that expression of the toxin gene is suppressed) without cleaving the off-target (with the result that the antibiotic resistance gene is expressed). Therefore, cells into which a target-specific nuclease with high specificity is introduced and in which only the target is cleaved and the off-target is not cleaved can survive treatment with an antibiotic. Thus, target-specific nucleases with high specificity or activity can be screened based on whether these selection elements are expressed.

As yet another example, when a target-specific nuclease is screened by using a mid-target comprising a toxin gene as a selection element and an off-target comprising a gene encoding a fluorescent protein as a selection element, if a vector comprising the mid-target and a vector comprising the target-specific nuclease are introduced into a cell, it can be expected that the target-specific nuclease exhibiting high specificity cleaves the mid-target (as a result of which expression of the toxin gene is inhibited) without cleaving the off-target (as a result of which the fluorescent protein is expressed). Therefore, cells into which a target-specific nuclease with high specificity is introduced and in which only the target is cleaved but the off-target is not cleaved can survive, and can be distinguished using a fluorescent protein.

Thus, the selection element comprised in the mid-target and/or off-target targets can be a reference for determining and selecting high specificity or high activity of target-specific nucleases.

4. Method for selecting nuclease

A selection system can be used to select for target-specific nucleases with high specificity or activity.

As one aspect, the invention relates to a method of selecting target-specific nucleases having high specificity or high activity in a plurality of target-specific nuclease test sets.

In the present invention, "a method of selecting a target-specific nuclease/a method of selecting a target-specific nuclease" includes a composition of selecting a cell containing a target-specific nuclease having high specificity or activity and then recognizing a specific nuclease. For example, excellent sequence information of target-specific nucleases can be obtained by the present invention.

First embodiment of 4-1 selection method

As a specific embodiment of the method of the present invention,

the method is useful as a method for selecting a target-specific nuclease having high specificity and high activity from a variety of target-specific nuclease "variants".

The method is a method for selecting a target-specific nuclease having high specificity or high activity, using the following components:

ii) a target-specific nuclease variant,

the method comprises the following steps:

a) Introducing component i) and component ii) into a cell;

c) Selecting the nuclease contained in the cells sorted in b) as a target-specific nuclease having high specificity or high activity.

4-1-1 component

The target-specific nuclease of component ii) can be a CRISPR-Cas system and can be a ZFN, TALEN, fokI, etc., furthermore, without being limited thereto.

The CRISPR-Cas system may consist of a guide RNA and a CRISPR enzyme.

The target-specific nuclease variant of component ii) may be a CRISPR-Cas system variant, preferably may be a CRISPR enzyme variant, but is not limited thereto. Nuclease variants can be produced using electromagnetic waves, UV, radiation, chemicals, exogenous/endogenous gene action, and the like. In one example, the variants are obtained by irradiating the WT CRISPR-Cas system with UV.

The Cas9 variant may be a variant in which the on-target effect is increased or decreased. In this case, the on-target effect means that the on-target position is cleaved or modified by Cas9.

The Cas9 variant may be a variant in which off-target effects are increased or decreased. In this case, off-target effects mean that the off-target position is cleaved or modified by Cas9.

Component ii) may use a vector system, and when component ii) is a CRISPR-Cas system, the guide RNA and the CRISPR enzyme may be comprised in the same vector or in different vectors.

Component ii) may be artificially synthesized.

4-1-2 Process

The cell may be a prokaryotic cell or a eukaryotic cell.

In addition, off-target targets may be present in the genome of the cell.

In addition, the on-target may be present in the genome of the cell.

In this case, in b),

among the cells into which components i) and ii) have been introduced in a), cells can be selected in which only the targeted targets in component i) are cleaved by component ii).

In addition, cells in component i) can be selected which are not cleaved by component ii) for off-target targets.

For example, when the off-target comprises an antibiotic resistance gene, cells that are antibiotic resistant can be selected in the presence of an antibiotic by allowing the antibiotic resistance gene to be expressed because the off-target is not cleaved.

Alternatively, when the off-target is located in the genome of the cell, the genome of the cell is normally retained as it is not cleaved by component ii), with the result that the cell is able to survive.

In this case, the cells sorted in b) may be cells in which the in-target of component i) is cleaved by component ii) and the off-target is not cleaved by component ii). These cells can be distinguished by means of a selection element.

In this case, in c),

the cells sorted in b) can be used to recognize component ii), i.e., the target-specific nuclease, introduced into the sorted cells.

Component ii) introduced into the sorted cells, i.e. the target-specific nuclease (using the sorted cells in b)) may be a target-specific nuclease capable of selectively cleaving or modifying only the target in question.

Obtaining the sequence of the nuclease by sequencing the nuclease obtained from the cells sorted in c), thereby obtaining a target-specific nuclease with high specificity or activity.

For example, when colonies were grown to a size that could be seen with the naked eye by culturing electroporated E.coli on LB agar plates containing kanamycin, chloramphenicol, and arabinose at 30 ℃ for 16 hours, each colony was inoculated into 10ml of LB medium containing chloramphenicol, followed by culture at 42 ℃ for 12 hours in a shaker. After the E.coli pellet was obtained by centrifugation, the plasmid was extracted from the pellet using a miniprep kit. The extracted plasmids can be sequenced using, for example, sanger sequencing methods. For the primers, a universal sequencing primer (e.g., CMV-F or BGH-R) existing outside of the N-and C-termini of Cas9 and a sequencing primer of the middle portion of Cas9 can be used.

As a specific embodiment of the present invention, when described with reference to FIG. 1, in the method of recognizing a target-specific nuclease, plasmid A, plasmid B and plasmid C were all introduced by electroporation on LB agar plates containing kanamycin and chloramphenicol, which are recognition markers for plasmid B and plasmid C, and arabinose (an arabinose-manipulating promoter), and when E.coli incubated for 1 hour in SOC containing anhydrotetracycline was divided into small portions, plasmid A was cleaved by the nuclease of plasmid C, and only E.coli in which genomic DNA was not cleaved survived.

Thus, target-specific nucleases with high specificity or activity can be selected by means of cells sorted by using the method described above.

4-2 second embodiment of the selection method

As a further specific embodiment, it is possible to,

the method can be used as a method for selecting a target-specific nuclease having high specificity and high activity in a target-specific nuclease "library".

ii) a library of target-specific nucleases,

the method comprises the following steps:

a) Introducing component i) and component ii) into a cell;

b) Sorting cells modified for only the target in a); and

c) Identifying component ii) using the cells selected in b).

4-2-1 component

The target-specific nuclease library can be a CRISPR-Cas system library. The nuclease libraries can be prepared directly, can be commercially available, and can be obtained from databases

The CRISPR-Cas system may consist of guide RNA and CRISPR enzyme.

Component ii) may be artificially synthesized.

In component i), the off-target may be a sequence or location of a gene or nucleic acid that forms one or more non-complementary binding to the guide RNA or guide sequence of the guide RNA of component ii).

4-2-2 Process

The cell may be a prokaryotic cell or a eukaryotic cell.

In addition, off-target targets may be present in the genome of the cell.

In addition, the on-target may be present in the genome of the cell.

In the step (b) of the above-mentioned process,

In this case, the cells sorted in b) may be cells in which the targeted target of component i) is cleaved by component ii) and the off-target is not cleaved by component ii). These cells can be distinguished by means of a selection element.

In this case, in c),

For example, when colonies were grown to a size that could be seen with the naked eye by culturing electroporated E.coli on LB agar plates containing kanamycin, chloramphenicol, and arabinose at 30 ℃ for 16 hours, each colony was inoculated into 10ml of LB medium containing chloramphenicol, followed by culture at 42 ℃ for 12 hours in a shaker. After the E.coli pellet was obtained by centrifugation, the plasmid was extracted from the pellet using a miniprep kit. The extracted plasmids can be sequenced using, for example, sanger sequencing methods. For the primers, universal sequencing primers (e.g., CMV-F or BGH-R) present outside the N-and C-termini of Cas9 can be used, as well as sequencing primers of the middle portion of Cas9.

Thus, target-specific nucleases with high specificity or activity can be selected in a target-specific nuclease library by means of cells sorted using the method described above.

5. Composition or kit

Another aspect of the present invention relates to a screening kit for selecting a target-specific nuclease with high specificity or high activity, the screening kit comprising: a multiplex target comprising one or more selection elements. The kit may be in the form of a composition.

In one embodiment, the composition or kit may consist of:

i) A multiplex target comprising one or more selection elements; and

ii) a target-specific nuclease and/or iii) a host cell.

The screening kit or composition for selection of target-specific nucleases with high specificity or high activity according to the present invention comprises a multiplex target comprising one or more selection elements as an essential component and optionally a target-specific nuclease and/or a host cell.

If desired, the kits of the invention can be provided as a 1-fluid kit comprising multiple targets comprising one or more selection elements.

If desired, the kits of the invention can be provided as 2-pack kits comprising multiple targets and host cells, respectively, containing one or more selection elements.

In this case, the kit can also be provided as a 1-liquid type by including multiple targets containing selection elements in the host cell. For example, the kit may be contained in the host cell as a separate plasmid or may be provided by integration into the host cell genome.

If desired, the kits of the invention can be provided as 3-fluid kits comprising, respectively, multiple targets comprising one or more selection elements, a host cell and a test nuclease.

In this case, the kit may also be provided as a 2-fluid type by including multiple targets containing selection elements in the host cell. For example, the kit may be contained in the host cell as a separate plasmid or may be provided by integration into the host cell genome.

In this case, component i) may use a vector system, and in component i) one or more on-target targets and one or more off-target targets may be contained in the respective vectors.

The target-specific nuclease of component ii) may be a CRISPR-Cas system, and may be ZFN, TALEN, fokI, etc., furthermore, is not limited thereto.

The CRISPR-Cas system may consist of a guide RNA and a CRISPR enzyme.

Component ii) may be artificially synthesized.

In component i), the on-target may be a sequence or position of a gene or nucleic acid that binds complementarily to the guide RNA or to the guide sequence of the guide RNA of component ii).

In component i), the on-target and/or off-target targets can be linked to a selection element.

Component iii) the host cell may be any cell suitable for expression of the selection element. Component iii) the host cell may be a prokaryotic cell or a eukaryotic cell.

For example, when the toxin gene ccdB is used, escherichia coli can be used as a host; when Hypoxanthine Phosphoribosyltransferase (HPRT) is used, cells derived from mammals can be used as host cells; when the toxin gene URA3 is used, yeast can be used as a host.

Furthermore, it is obvious that one skilled in the art of the present invention can arbitrarily select and use a cell type suitable for the screening and selection of nuclease.

Furthermore, if necessary, the composition or kit may further comprise a buffer for selection and various substances (e.g., antibiotics, X-gal, etc.) or various reagents for introduction into cells (e.g., transfection agent, liposome (lipofectamine), etc.).

6. Cell (line) or method for producing cell

In addition, the invention provides cells comprising and/or methods of producing a plurality of targets for screening and/or selecting target-specific nucleases.

Characteristics of the cells consisting in containing multiple targets.

In this case, the multiple targets may be multiple targets present in the genome of the cell, or may be multiple targets introduced into the cell from an external source.

The cell may be a prokaryotic cell or a eukaryotic cell.

6-1 multiple targets

The multiplexed targets may consist of one or more on-target targets and one or more off-target targets.

6-1-1 target

In this case, the intermediate target may be located in the genome of the cell.

In this case, the on-target may comprise a selection element.

The selection element may not be expressed when the target-in-target comprising the selection element is cleaved or modified by a target-specific nuclease.

6-1-2 off-target

When the target-specific nuclease is a CRISPR-Cas system, the off-target can comprise a PAM sequence that is recognized by the CRISPR enzyme.

An off-target can be a sequence or location of a gene or nucleic acid that comprises less than 100% complementary binding to the nucleic acid sequence of the portion of the target-specific nuclease that specifically recognizes the target.

In this case, the off-target may be located in the genome of the cell.

The off-target may additionally comprise a selection element.

The cell may comprise multiple targets diversely designed according to the target of the target-specific nuclease.

Furthermore, the cell may comprise only some of the multiple targets.

That is, in a multiplex target, a cell may contain only a medium target or only an off-target. 6-2 production method

Methods of producing cells may use methods of introducing a vector system comprising multiple targets into a cell.

In addition, for the introduction of multiple targets into a cell, multiple targets may be introduced simultaneously.

That is, a vector comprising an in-target and a vector comprising an off-target (these targets are the multiple targets) can be introduced into a cell simultaneously.

For the introduction of multiple targets into a cell, multiple targets may be introduced separately.

That is, in a multiplex target, a vector comprising an in-target may be introduced into a cell before a vector comprising an out-of-target may be introduced into a cell. Alternatively, in a multiplex target, the vector comprising the off-target may be introduced into the cell prior to introduction of the vector comprising the on-target into the cell.

Furthermore, for the introduction of multiple targets into a cell, only some of the multiple targets may be introduced.

That is, only a vector containing a medium target among multiple targets may be introduced into a cell. Alternatively, only a vector comprising an off-target of a multiplex of targets may be introduced into the cell.

The system of the invention can be advantageously used to select target-specific nucleases with high specificity and activity from any nuclease test set. In addition, off-target effects can be reduced and on-target effects can be increased by using the nucleases selected by the system of the invention when a gene is modified or manipulated.

Examples

The present invention will be described more specifically by way of examples.

These examples are only for illustrating the present invention, and it is apparent to those of ordinary skill in the art that the scope of the present invention should not be construed as being limited by these examples.

Example 1 screening of Cas9 variants in e.coli

1-1 control test

CATCAACATCGAATACATGA NAG (SEQ ID No. 1) is a repeat sequence found in K12 E.coli genomic DNA. This sequence comprises the PAM sequence NAG, similar to NGG, and is therefore known to be cleaved less efficiently by the CRISPR-Cas9 enzyme.

The 20-mer leader sequence CATCAACATCGAATACATGA (SEQ ID No. 2) from the above sequence was used as sgRNA in plasmid B. A 23-mer target sequence CATCAACATCGAATACATGA TGG (SEQ ID No. 3) targeted by sgRNA was inserted into plasmid a as a target site. This target sequence has NGG (but not NAG) as a PAM site, which is expected to exhibit high cleavage efficiency.

Plasmid A and plasmid B were both introduced into E.coli BW25141 using electroporation. The cells into which plasmid A and plasmid B were introduced were cultured in LB medium containing ampicillin and kanamycin antibiotics at 37 ℃. After growth of the colonies by cell culture, electroporation competent cells (electroporation cells) were prepared from the colonies using standard methods.

Plasmid C containing WT CAS9 as a positive control or an empty vector (null vector) as a negative control was introduced into the electroporation competent cells containing plasmid a and plasmid B. After culturing the cells in SOC medium for 1 hour, half of the cultured cells were cultured in LB medium containing chloramphenicol, and the other half of the cultured cells were cultured in LB medium containing chloramphenicol, kanamycin, 10mM arabinose, and 100ng/ml Anhydrotetracycline (ATC).

As a result, about 10,000 colonies were recognized in LB medium containing chloramphenicol for all cells into which plasmid C and an empty vector were introduced, respectively; no colonies were found in LB medium containing chloramphenicol, kanamycin, arabinose and anhydrotetracycline. The results are explained below.

In the cells introduced with plasmid C containing WT CAS9, since the expressed WT CAS9 has residual activity on the replaced PAM sequence NAG, the genomic DNA sequence of E.coli containing NAG was cleaved. On the other hand, in the cells into which plasmid C (i.e., empty vector) containing no substance was introduced, since plasmid a containing the toxin gene could not be cleaved due to lack of cleavage activity, expression of the toxin gene was induced in LB medium containing arabinose, thereby exhibiting apoptosis.

For another control, plasmid a 'and plasmid B' were used as positive controls, which contained sequences ctagatgagaccggatccggtctccTGG (SEQ ID No. 4) and ctagatgagaccggatccggtctcc (SEQ ID No. 5), respectively, which were not present in the genomic DNA of e. After plasmid C containing WT CAS9 was introduced into it, about 10,000 colonies were observed in each of LB medium containing chloramphenicol only and LB medium containing chloramphenicol, kanamycin, arabinose, and anhydrotetracycline. This is believed to be a result of the WT CAS9 cleaving plasmid a efficiently without destroying genomic DNA. 1-2 mutations

After a successful control experiment, random mutations were introduced to the sequence of WT CAS9 by PCR amplification using the Genemorph II kit manufactured by Agilent Technologies, inc. The PCR product was ligated into the backbone of the double digested plasmid C. A library of 1,000,000 size was constructed by introducing the plasmid generated by ligation into high-efficiency electroporation competent cells. Random libraries were introduced into electroporation competent cells containing plasmid a and plasmid B previously prepared.

Subsequently, the cells were cultured in both media in the same manner as in the previous experiment. As a result, about 2,500 colonies were recognized in LB medium containing only chloramphenicol, whereas 180 colonies were revealed in LB medium containing chloramphenicol, kanamycin, arabinose, and anhydrotetracycline. Each colony was cultured in LB medium containing chloramphenicol to allow expression of plasmid C containing random mutations. It was confirmed that all sequences of random mutations obtained from the cultured colonies were effective mutations having activities against the universal PAM sequence NGG, but exhibiting suppressed activities against NAG (which is a similar but not universal PAM sequence).

Through this experiment, cas9 variants specific for the guide sequence can be found. In this case, the mismatched sequence of the sgRNA sequence may be an endogenous genomic DNA sequence of e.coli or an artificial sequence introduced and inserted from an external source into e.coli.

By using the screening methods described above, a number of Cas9 variants were successfully identified that had reduced activity against the universal PAM sequence NGG, but also against NAG (which is a similar but not universal PAM sequence).

Example 2: selection of nucleases

It was confirmed that 20-mer sgrnas having 1 mismatch compared to the guide sequence of the target position in the WT EMX1 gene had reduced activity compared to the case where sgrnas without mismatches (i.e., perfect matches) were used in the previous studies. The WT EMX1 gene is present in the genomic DNA of mammalian cells, and a PCR product obtained by amplifying a 500-mer base sequence including the WT EMX1 gene in the genomic DNA of mammalian cells is inserted into the genomic DNA of Escherichia coli (BW 25141) by using a standard method (McKenzie GJ et al, BMC Microbiol.2006,6, 39). As a result, the resulting strain was named BW25141-EMX1 and used thereafter.

20-mer leader sequence comprising one or more mismatches with respect to the target sequence gagtccgagcagaagaagaaggg (SEQ ID No. 6):

in an embodiment of the invention, the following sequences were used as sgrnas in plasmid B:

56：gagtccgagcagaaAGagaa(SEQ ID No.7)；

1718：gaACccgagcagaagaagaa(SEQ ID No.8)；

7: gagtccgagcagaGgaagaa (SEQ ID No. 9); and

17：gagCccgagcagaagaagaa(SEQ ID No.10)。

the 23-mer target sequences corresponding to the sgrnas described above, namely 56 (gagtccgagcagaaAGagaa ggg), 1718 (gaACccgagcagaagaagaa ggg), 7 (gagtccgagcagaGgaagaa ggg), 17 (gagCccgagcagaagaagaa ggg), were inserted into plasmid a as target positions.

Both plasmid A and plasmid B produced as described above were introduced into E.coli strain BW25141-EMX1 using electroporation. The cells into which plasmid A and plasmid B were introduced were cultured in LB medium containing ampicillin and kanamycin antibiotics at 37 ℃. After colony culture, electroporation competent cells were prepared using standard protocols. After growth of the colonies by cell culture, electroporation competent cells were prepared from the colonies using standard methods.

i) Control reactions of 56 and 1718

For control reactions, plasmid C containing WT CAS9 or an empty vector as a negative control was introduced into electroporation competent cells containing plasmid a and plasmid B. After culturing the plasmid C-introduced or empty vector-introduced cells in SOC medium containing 1,000ng/ml anhydrotetracycline for 1 hour, half of the cultured cells were cultured in LB medium containing chloramphenicol, and the other half of the cultured cells were cultured in LB medium containing chloramphenicol, kanamycin, 10mM arabinose and 1,000ng/ml anhydrotetracycline.

The results obtained by performing a control reaction of plasmid B containing two mismatched sequences compared to the EMX-1 sequence inserted into the genomic DNA of E.coli (i.e., plasmid B containing 56 and plasmid B containing 1718) are to show a comparison between the number of colonies formed per unit in LB agar medium (CK) containing chloramphenicol and kanamycin and the number of colonies formed per unit in LB agar medium (CKA-1, 000ng/ml ATC) containing chloramphenicol, kanamycin, arabinose and 1,000ng/ml anhydrotetracycline (FIG. 2).

When two mismatches are located near the seed region (56-WT-CAS 9), less than 0.1% of Cas 9-introduced E.coli survived. On the other hand, when an empty vector (instead of Cas 9) as a negative control was introduced into e.coli, about 0.01% of e.coli survived. The results of the negative control were considered as background due to the effect of plasmid A not functioning.

ii) control reactions of 7 and 17

For control reactions, plasmid C containing WT CAS9 or an empty vector as a negative control was introduced into electroporation competent cells containing plasmid a and plasmid B. After culturing the cells into which plasmid C or the empty vector was introduced in SOC medium containing 10ng/ml anhydrotetracycline for 1 hour, half of the cultured cells were cultured in LB medium containing chloramphenicol, and the other half of the cultured cells were cultured in LB medium containing chloramphenicol, kanamycin, 10mM arabinose, and 10ng/ml anhydrotetracycline.

The results obtained by performing a control reaction of plasmid B containing one mismatch sequence compared to the EMX-1 sequence inserted into the E.coli genomic DNA (i.e., plasmid B containing 7 and plasmid B containing 17) are shown in comparison between the number of colonies formed per unit in LB agar medium (CK) containing chloramphenicol and kanamycin and the number of colonies formed per unit in LB agar medium (CKA-10 ng/ml ATC) containing chloramphenicol, kanamycin, arabinose, and 10ng/ml anhydrotetracycline (FIG. 3).

When a mismatch is located near the seed region (7-WT-CAS 9), less than 0.1% of Cas 9-introduced E.coli survives. On the other hand, when an empty vector (instead of Cas 9) as a negative control was introduced into e.coli, about 0.01% of e.coli survived. The results of the negative control were considered as background due to the effect of plasmid A not functioning.

iii) Screening of 56 and 1718

After a successful control experiment, random mutations were introduced in three different ways.

1. PCR amplification of WT CAS9 sequences was performed using genemorphh II kit manufactured by Agilent Technologies, inc.

A diversification PCR random mutagenesis kit manufactured by Clontech laboratories, inc.

3. Coli strains (XL 1-red) producing variants of Agilent using standard experimental methods with low error rates.

Each PCR product obtained by the above method was ligated into the backbone of plasmid C, which was double digested. By introducing the plasmids generated by ligation into highly competent electroporation competent cells, libraries each having a size of 2,000,000 were constructed. Each random library was introduced into electroporation competent cells prepared using previously prepared BW25141-EMX1 comprising plasmid A (comprising 56) and plasmid B (comprising 56).

The cells into which each random library was introduced were cultured and formed into colonies in a medium (CKA) containing chloramphenicol, kanamycin, and arabinose, and an integrated library (integrated library) was formed by counting the number of formed colonies and collecting the formed colonies.

The obtained library was cultured in LB medium containing chloramphenicol at 42 ℃, thereby selectively removing sgRNA vectors containing a vector having a temperature sensitive origin (origin). After culturing, the library was purified using miniprep (miniprep) to obtain a library vector.

For the library vector obtained by purification, the same experiment was performed using BW25141-EMX1 containing plasmid a (containing 1718) and plasmid B (containing 1718) in the same manner as described above to perform another selection.

The selection of library vectors is performed continuously until the ratio of the number of CKA/CK colonies reaches 100%. iv) shuffling reaction (shuffling reaction)

The libraries obtained via selection were used in the shuffling reaction by using standard shuffling methods including additional modifications. It is known that shuffling efficiency is significantly reduced when the PCR product is typically greater than 1 kb. Thus, shuffling reactions do not readily occur for Cas9 vectors that are 4,300 base pairs in length.

The Cas9 vector was divided into 5 fragments and each PCR fragment was shuffled using standard methods. The shuffling mixture is then brought together and the PCR reaction is performed again. The PCR product thus obtained was amplified using primers targeting the 5 'and 3' ends of Cas9, and generation of the shuffling library was confirmed from the amplified PCR product by recognizing the size of the product by means of agarose gel. v) screening of 7 and 17

The random mutant library and shuffled library prepared using the Agilent and Clontech kit were screened repeatedly by using EMX1-BW25141 containing plasmid a (containing 7 or 17) and plasmid B (containing 7 or 17) in a manner similar to the screening reaction using 56 and 1718. The screening reaction was repeated until a stable ratio was reached. As a result, the colonies obtained were analyzed by using standard sequencing methods.

vi) verification

On-target and off-target experiments were performed for the DMD gene and the EMX1 gene, respectively, by using a complete library obtained by performing both methods simultaneously (shuffling + 1-mismatch (7 or 17) screening by 1-mismatch (7 or 17) and 2-mismatch (56 or 1718) screening).

As a result, when screening was started from a 2-mismatch (56 or 1718), it could be confirmed that the off-target effect was more significantly reduced than that of the WT Cas9 (fig. 6 and 7).

Experiments to identify specific improvements in mammalian cells were performed using 3 clones selected via screening. On-target and off-target experiments were performed for the DMD gene and the EMX1 gene, respectively.

As a result of determining the on-target and off-target activities of the DMD gene by using the complete library obtained by screening by simultaneously performing both methods, it can be confirmed that the off-target activity is more significantly reduced compared to the WT Cas9 when screening is started from two mismatches (fig. 8).

As a result of determining the on-target and off-target activities of the EMX1 gene by using the complete library obtained by screening by simultaneously performing both methods, it can be confirmed that the off-target activity is more significantly reduced compared to the WT Cas9 when screening is started from two mismatches (fig. 9).

From these results, it was confirmed that the specificity of both genes was improved compared to WT-spCas 9.

Example 3: screening of mammalian cells

The experiments described in examples 1 and 2 can be carried out using mammalian cells.

The HPRT gene can be used similarly to the toxin gene ccdB. gRNA sequences with mismatches to HPRT bind to the 5' end of thymidine kinase and cysteine deaminase (after the start codon), the full sequence comprising a selectable marker (e.g., zeocin or puromycin) is inserted into mammalian cells (e.g., HELA cells) by using standard methods, and stable cell lines can be prepared by sorting the cells thus obtained using antibiotics.

A vector comprising the Cas9 variant can be inserted into the genomic DNA of the cell line using standard methods by using transposons (in this case comprising transposases and can be, for example, piggybac, sleepingbeauty, etc.) or viruses (lentiviruses). sgRNA vectors targeting HPRT can be transfected into this cell line and HPRT and TK or CD can each be selected by using 6-thioguanine and ganciclovir or 5-fluorocytosine.

The colonies thus selected contain Cas9 variants with improved specificity. The screening method can be used without TK or CD and sgrnas showing negligible activity can be used to find Cas9 with increased activity.

Industrial applicability

Turkin <110> K.K

<120> method for screening target-specific nuclease using multiple target system of on-target and off-target and use thereof

<130> OPP17-033-PCT

<150> PCT/US 62/350,282

<151> 2016-06-15

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Claims

1. A method for assessing the specificity or activity of a CRISPR enzyme, the method comprising:

providing at least one E.coli cell comprising a first plasmid and a genome,

wherein the first plasmid comprises a first nucleic acid and a first selection element operably linked to the first nucleic acid,

wherein the first selection element is a gene selected from the group consisting of a toxin gene, an antibiotic resistance gene, a gene encoding a fluorescent protein, a lacZ gene, and a gene encoding luciferase,

wherein the genome comprises a second nucleic acid having less than 100% sequence homology to the first nucleic acid;

contacting a CRISPR complex with the first nucleic acid and the second nucleic acid, respectively,

wherein the CRISPR complex comprises the CRISPR enzyme and a guide RNA capable of forming a CRISPR complex with the CRISPR enzyme,

wherein the CRISPR enzyme is a Cas9 protein or a Cpf1 protein,

wherein the first nucleic acid is an on-target of the CRISPR complex,

and, the second nucleic acid is an off-target of the CRISPR complex;

the identification was carried out for each E.coli cell,

i) Whether or not said first selection element is expressed, and

ii) whether the E.coli cell is dead or not,

thereby enabling the identification of whether off-target and on-target modifications have occurred in the cell; and

assessing the specificity or activity of the CRISPR enzyme based on the result of the identifying.

2. The method of claim 1, wherein the first selection element is a ccdB gene.

3. The method of claim 1, wherein said CRISPR enzyme is a non-naturally occurring mutant CRISPR enzyme.

4. The method of claim 1, wherein the CRISPR enzyme is Cas9.

5. The method of claim 4, wherein the Cas9 is selected from the group consisting of:

streptococcus pyogenes Cas9; staphylococcus aureus Cas9; streptococcus thermophilus Cas9; neisseria meningitidis Cas9; campylobacter jejuni Cas9; and their orthologues.

6. An e.coli cell for assessing the specificity or activity of a CRISPR complex, the e.coli cell comprising:

a first plasmid comprising a first nucleic acid and a first selection element operably linked to the first nucleic acid,

wherein the first selection element is a gene selected from the group consisting of a toxin gene, an antibiotic resistance gene, a gene encoding a fluorescent protein, a lacZ gene, and a gene encoding luciferase; and

a genome comprising a second nucleic acid having less than 100% sequence homology to the first nucleic acid,

wherein the first nucleic acid is an on-target of the CRISPR complex,

wherein the second nucleic acid is an off-target of the CRISPR complex,

wherein the second nucleic acid is exogenous, not endogenous, in the cell.

7. The Escherichia coli cell of claim 6, wherein the first selection element is a ccdB gene.

8. A method for assessing the specificity or activity of a CRISPR enzyme, the method comprising:

providing at least one cell comprising a first plasmid and a second plasmid,

wherein the second plasmid comprises a second nucleic acid having less than 100% sequence homology to the first nucleic acid and a second selection element operably linked to the second nucleic acid,

wherein the first selection element and the second selection element are each independently selected from the group consisting of a toxin gene, an antibiotic resistance gene, a gene encoding a fluorescent protein, a lacZ gene, and a gene encoding luciferase,

wherein the first selection element and the second selection element are different from each other;

wherein the CRISPR enzyme is a Cas9 protein or a Cpf1 protein,

wherein the first nucleic acid is an on-target of the CRISPR complex,

wherein the second nucleic acid is an off-target of the CRISPR complex;

the identification is carried out for each cell,

whether or not said first selection element is expressed, and

whether or not said second selection element is expressed,

thereby enabling identification of whether off-target and on-target modifications have occurred in the cell; and

assessing specificity or activity of the CRISPR enzyme based on the result of the recognition.

9. The method of claim 8, wherein the first selection element is a ccdB gene.

10. The method of claim 8, wherein said CRISPR enzyme is a non-naturally occurring mutant CRISPR enzyme.

11. The method of claim 8, wherein the CRISPR enzyme is Cas9.

12. The method of claim 11, wherein the Cas9 is selected from the group consisting of:

13. A cell for assessing the specificity or activity of a CRISPR complex, the cell comprising:

a first plasmid comprising a first nucleic acid and a first selection element operably linked to the first nucleic acid; and

a second plasmid comprising a second nucleic acid having less than 100% sequence homology to the first nucleic acid and a second selection element operably linked to the second nucleic acid,

wherein the first nucleic acid is an in-target of the CRISPR complex,

wherein the second nucleic acid is an off-target of the CRISPR complex,

wherein the first selection element and the second selection element are genes independently selected from the group consisting of a toxin gene, an antibiotic resistance gene, a gene encoding a fluorescent protein, a lacZ gene, and a gene encoding luciferase,

wherein the first selection element and the second selection element are different from each other.

14. The cell of claim 13, wherein the first selection element is a ccdB gene.

15. A kit for assessing the specificity or activity of a CRISPR enzyme comprising the cell of claim 6 or 13.